university  of  ILLINOIS  LIBRARY 

APR  i 0 1917 


THE  RELATIONS  OF  BURSARIA 
TO  FOOD 

I.  SELECTION  IN  FEEDING  AND  IN 
EXTRUSION 


BY 

ELMER  J.  LUND 


A DISSERTATION  SUBMITTED  TO  THE  BOARD  OF 
UNIVERSITY  STUDIES  OF  THE  JOHNS 
HOPKINS  UNIVERSITY 

IN  CONFORMITY  WITH  THE  REQUIREMENTS  FOR 
THE  DEGREE  OF  DOCTOR  OF 
PHILOSOPHY 


SUBMITTED  JANUARY  2,  191*^ 


Reprinted  from  The  Journal  of  Experimental  Zoology,  Vol.  i6,  No.  i 
January,  1914 


ACKNOWLEDGMENTS 


I wish  to  express  my  indebtedness  especially  to  Professors  H.  S. 
Jennings,  E.  A.  Andrews,  D.  S.  Johnson  and  B.  E.  Livingston, 
for  the  stimulating  intellectual  discipline  which  contact  with  them 
has  given  me.  I am  also  deeply  grateful  to  the  Electors  of  the 
Adam  T.  Bruce  Fellowship  for  designating  me  as  a holder  of  that 
Fellowship. 


Reprinted  from  The  Journal  of  Experimental  Zoology,  Vol.  16,  No.  1 
January,  1914 


THE  RELATIONS  OF  BURSARIA  TO  FOOD 

I.  SELECTION  IN  FEEDING  AND  IN  EXTRUSION 
E.  J.  LUND 

Zoological  Laboratory  of  The  Johns  Hopkins  University 

EIGHT  FIGURES 

CONTENTS 

Introduction : 2 

Action  of  the  structural  mechanism  for  feeding  and  the  selection  of  food ...  4 

1.  Rejection 5 

2.  Acceptance 5 

Formation  of  the  vacuole  and  elimination  of  residues 7 

Measurement  of  the  amount  of  food  eaten,  and  method  of  experimentation. . 8 

Internal  relations  affecting  the  feeding  process 10 

1.  The  relation  of  the  physiological  state  of  the  organism  to  the  feeding 

process 10 

2.  Changes  in  the  physiological  state  as  shown  by  using  the  feeding  proc- 
ess as  an  index 16 

3.  Other  causes  of  individual  variation 19 

The  external  relations  of  the  feeding  process 20 

1.  Effects  of  external  factors  on  feeding 20 

a.  Concentration  of  the  food  supply 20 

b.  Effect  of  mechanical  stimulation  and  mechanical  injury  on  feeding.  21 

c.  Temperature 23 

d.  HCl  and  NaOH 24 

e.  White  light 24 

f.  The  electric  current 26 

Selection  of  food  and  the  factors  concerned 29 

1.  Experiments  with  stained  and  unstained  yolk 30 

a.  Saffranin 30 

b.  Janus  green 34 

c.  Hematoxylin ' 37 

d.  Congo  red 39 

e.  Sudan  III 40 

f.  Stale  yolk 40 

2.  The  basis  for  and  the  nature  of  the  selection  of  food  in  Bursaria 41 

The  relation  of  Bursaria  to  digestible  and  non-digestible  substances 43 

1.  External  relations 43 

2.  Internal  relations 44 

Summary 51 

Literature  cited 52 

1 

THE  JOURNAL  OF  EXPERIMENTAL  ZOOLOGT,  VOL.  16,  NO.  1 
JANUARY,  1914 


2 


E.  J.  LUND 


INTRODUCTION 

While  studying  the  phenomena  of  regeneration  and  structural 
regulation  in  Bursaria,  it  became  desirable  to  know  something 
of  the  relations  of  this  organism  to  its  food  and  of  the  processes 
which  the  solid  food  material  undergoes  in  its  passage  into  and 
through  the  cell  and  in  the  elimination  of  residues.  This  paper 
aims  to  present  the  first  part  of  these  observations  and  to  make 
a general  survey  of  the  relations  of  this  organism  to  food,  leading 
up  to  a more  detailed  study  of  these  and  certain  other  problems 
of  the  cell  as  found  in  this  unicellular  animal. 

Bursaria  was  found  to  be  much  more  favorable  for  the  investi- 
gation of  these  phenomena  than  the  smaller  infusoria  such  as 
Paramecium.  No  study  has  heretofore  been  made  of  the  rela- 
tions of  Bursaria  to  food,  so  that  the  facts  herein  presented  are 
new.  The  present  investigation  attempts  not  only  to  determine 
qualitatively  whether  the  relations  to  food  are  similar  to  those 
more  or  less  known  for  Paramecium,  Stentor,  Vorticella  and 
other  infusoria,  but  more  particularly  to  work  out  and  express 
these  relations  in  a more  quantitative  way  than  has  been  done 
heretofore.  It  was  found  that  certain  kinds  of  experimental  tests 
such  as  those  on  the  rate  of  digestion,  could  be  made  upon  this 
form,  which  it  would  have  been  difficult  or  impossible  to  carry 
out  on  smaller  unicellular  organisms.  Its  very  large  size  offers 
a singular  opportunity  for  easy  manipulation  in  many  kinds  of 
work.  When  in  a clear  medium  it  is  readily  visible  to  the  naked 
eye  at  a distance  of  six  or  eight  feet  and  individuals  may  be 
transferred  singly  with  a pipette  without  the  aid  of  any  magni- 
fying instruments. 

Bursaria  occurs  not  infrequently  in  cultures  brought  from 
ponds  in  the  vicinity  of  Baltimore,  though  it  is  less  common 
than  many  other  Protozoa.  It  can  readily  be  cultivated  in  large 
culture  dishes  in  the  laboratory.  In  this  way  I have  had  abun- 
dant material  at  my  disposal  for  many  months.  The  method 
of  cultivation  has  been  simply  the  inoculation  of  an  infusion  of 
timothy  hay  in  tap  water  from  the  wild  culture;  by  several  in- 
oculations at  different  times  one  usually  succeeds  in  obtaining 
large  numbers.  Since  the  food  of  this  organism  is  not  bacteria. 


RELATION  OF  BURSARIA  TO  FOOD 


3 


SO  far  as  I have  yet  observed,  but  a variety  of  other  ciliates, 
flagellates  and  rhizpods,  it  is  difficult  to  find  a culture  medium 
which  can  be  readily  manipulated,  and  hence  pure  line  cultures 
can  not  be  obtained  so  readily  as  of  a form  like  Paramecium. 
The  problem  of  pure  line  cultivation  of  this  organ  sm  will  not 
be  dealt  with  in  this  paper.  The  material  for  use  in  the  follow- 
ing experiments  has  all  been  obtained  from  mixed  or  Vild’  cul- 
tures, though  the  reinoculations  from  the  single  parent  culture 
brought  into  the  laboratory  seven  months  ago  resulted  in  a small 
number  of  pure  lines  living  side  by  side  in  the  cultures. 

It  is,  in  fact,  preferable  in  some  ways  to  use  material  from 
such  wild  cultures  for  the  kind  of  experiments  to  be  considered 
in  this  paper. 

Even  without  the  aid  of  pure  cultures  or  the  application  of 
statistical  methods  to  wild  cultures  it  soon  became  apparent 
that  there  are  actually  at  least  two  very  distinct  races  of  Bur- 
saria  which  differ  in  several  diverse  characters,  physiological  as 
well  as  morphological.^  One  form,  which  under  certain  food 
conditions  has  a tail,  has  been  used  exclusively  in  these  experi- 
ments, since  the  other  form,  collected  at  the  same  time  and 
lacking  a tail,  died  out  early  in  the  experiments. 

I have  observed  the  following  organisms  to  be  eaten  and  di- 
gested by  Bursaria:  Chilomonas,  Colpidium  colpoda,  Vorticella 
and  some  of  its  relatives,  Oxytricha,  Stylonychia,  Arcella,  Sten- 
tor,  Paramecium,  Stephanodiscus,  and  some  kinds  of  rotifers. 
Only  once  have  I observed  bacteria  to  be  eaten,  and  that  time 
in  the  form  of  zoogloea.  It  is,  however,  certain  that  bacteria 
form  only  a small  part,  if  any,  of  the  usual  diet  of  this  organism. 
The  smaller  ciliates,  flagellates  and  rhizopods  are  the  favorite 
article  of  food.  The  larger  organisms,  such  as  Stentor,  are  sel- 
dom successfully  captured.  Paramecium  is  quite  commonly 
eaten,  though  Bursaria  does  not  seem  to  thrive  well  on  this 
food.  Occasionally  rotifers  are  eaten  and  it  was  observed  on 
several  occasions  that  these  may  remain  alive  within  the  vacuole 

^ I have  been  unable  to  find  reference  in  the  literature  to  more  than  one  form 
of  Bursaria.  A consideration  of  the  problems  connected  with  the  existence  of 
diverse  ‘races’  of  Bursaria  will  be  left  for  a later  time. 


4 


E.  J.  LUND 


for  as  long  as  five  hours  before  they  are  killed.  It  is,  however, 
plainly  evident  when  one  follows  the  development  of  wild  cul- 
tures from  day  to  day  that  some  forms  are  eaten  in  greater  num- 
bers than  others  and  if  the  smaller  forms,  such  as  Colpidium, 
Vorticella  and  Arcella,  are  present  in  abundance  along  with  such 
forms  as  Paramecium,  Stentor  and  Stylonychia,  the  former  kinds 
serve  exclusively  as  food  for  Bursaria  while  the  latter  are  rarely 
eaten.  When  the  cycle  of  development  of  the  culture  comes  to 
the  stage  where,  for  example,  Paramecium  is  in  superabundance, 
then  the  body  of  Bursaria  may  be  more  or  less  filled  with  Para- 
mecia.  In  contrast  to  the  above  mentioned  forms,  Spirostomum 
ambiguum  was  always  rejected.  It  was  often  seen  to  be  taken 
into  the  oral  pouch  but  invariably  was  thrown  out  again,  while 
Paramecia  present  in  the  same  culture  were  readily  eaten  by  the 
same  Bursaria  individuals  at  the  time  of  the  observations.  This 
is  the  only  case  where  Bursaria  was  seen  definitely  to  discrimi- 
nate between  two  different  forms  of  Protozoa. 

By  simple  methods  of  observation  like  the  above,  it  would 
be  impossible  to  determine  just  what  the  principle  and  the  fac- 
tors are  that  determine  whether  Bursaria  will  feed  on  only  one 
or  several  or  all  of  these  forms  if  they  be  present  in  all  the  cul- 
tures simultaneously,  which  of  course  they  often  are.  It  is  with 
the  object  of  elucidating  these  and  certain  related  questions  that 
the  following  comparatively  simple  experiments  have  been  per- 
formed, by  limiting  and  determining  to  a high  degree  the  condi- 
tions under  which  this  organism  will  react  to  food. 

ACTION  OF  THE  STRUCTURAL  MECHANISM  FOR  FEEDING  AND 
THE  SELECTION  OF  FOODS 

An  account  of  the  food  relations  of  Bursaria  requires  us  to 
examine  in  some  detail  the  objective  processes  involved  in  feed- 
ing; these  are  very  striking.  The  highly  developed  oral  appa- 
ratus with  its  large  cilia,  when  in  operation  during  the  feeding 
process,  may  easily  be  observed.  When  the  organisms  are  fed 
on  such  substances  as  yolk  or  starch  they  usually  sooner  or 
later  become  quiet  for  a time,  and  settle  to  the  bottom  of  the 
dish  or  stick  to  the  surface  film  of  the  water,  then  they  may  be 


RELATION  OF  BURSARIA  TO  FOOD  5 

observed  under  a high  power  of  a binocular.  Granular  sub- 
stances of  different  chemical  or  physical  properties  may  be  placed 
in  the  medium  and  the  path  of  each  individual  particle  may  be 
easily  observed.  Mixtures  of  these  substances  may  also  be 
made  and  the  paths  of  the  different  kinds  of  particles  may  be 
determined. 

The  different  paths  of  particles  which  come  into  varying  rela- 
tions with  the  organism  are  shown  by  the  arrows  in  the  outline 
drawings  of  figure  1.  The  paths  of  the  arrows  are  correct  repre- 
sentations of  the  paths  taken  by  the  different  kinds  of  particles. 
In  general  the  paths  taken  by  particles  may  be  distinguished 
according  to  the  following  outline: 

1.  Paths  of  rejection 

a.  Path  of  total  rejection,  arrows  A 

h.  Path  of  rejection  of  larger  particles  taken  into  the  oral 
apparatus,  arrows  B 

c.  Paths  of  rejection  of  smaller  particles  taken  into  the  oral 
apparatus,  arrows  Ci  and  C2. 

These  paths  may  also  be  slightly  modified  by  a combination 
of  the  avoiding  reaction  with  the  different  rejection  reactions. 

2.  Path  of  acceptance  of  large  and  small  particles  {large  arrows  D) 

Path  A is  taken  by  those  .particles  which  under  conditions 
hereafter  to  be  considered  (p.  29)  never  enter  the  oral  apparatus 
and  are  only  drawn  towards  the  body  by  the  current;  for  exam- 
ple, very  toxic  particles  of  yolk.  Path  B is  always  taken  b3^ 
those  particles  which  are  too  large  to  pass  out  by  way  of  path 
Cl  and  C2  and  must  be  passed  back  to  the  exterior  by  the  same 
way  as  they  were  taken  in,  in  order  for  the  organism  to  get  rid 
of  them  at  all.  This  may  be  illustrated  by  the  larger  properly 
treated  grains  of  hard  boiled  yolk.  The  path  represented  b^^ 
the  arrows  Ci  and  C2  has  considerable  range  of  variation  in  part 
of  its  course.  It  may  be  illustrated  by  cornstarch  grains;  these 
are  of  convenient  size.  The  variations  in  the  course  of  these 
particles  may  be  divided  roughtl^"  into  two  main  divisions ; some 
follow  the  dotted  arrows  C2  and  never  directly  retrace  an}^ 


6 


E.  J.  LUND 


Fig.  1 /,  Outline  drawing  from  dorsal  side  of  Bursaria,  to  show  position  of 
the  oral  pouch  in  the  body,  and  the  paths  of  variously  rejected  and  accepted 
particles.  A,  path  of  total  rejection;  B,  path  of  rejection  of  large  particles  which 
are  too  large  to  pass  out  by  way  of  the  oral  sinus,  S.  Ci  and  paths  of  rejection 
of  small  particles;  these  pass  out  by  way  of  the  oral  sinus,  S;  D,  path  of  accept- 
ance. II,  Outline  drawing  of  sagittal  section,  in  the  plane  through  the  body 
represented  by  the  straight  line  through  7.  A,  path  of  total  rejection;  D,.  path 
of  entrance  of  all  particles  taken  into  the  oral  pouch,  same  as  first  part  of  path 
D,  I.  C,  path  of  rejection  of  small  particles,  same  as  Ci  and  C2  of  7.  E,  I the 
same  as  E 77,  direction  of  rejected  particles,  C,  7,  and  Ci,  C2,  77. 

part  of  their  former  path;  some  pass  up  into  the  proximal  end 
of  the  oral  pouch  but  are  rejected  and  returned  to  the  outside 
by  way  of  the  continuous  arrows  Ci.  All  the  paths  of  rejection 
under  Ci  and  C2  converge  and  lead  to  the  exterior  by  way  of 
the  oral  sinus,  figure  1,  I and  II  S.;  they  then  pass  backward 
under  the  posterior  ventral  side,  as  shown  by  arrows  E.  There 
is  but  one  path  of  acceptance  for  both  large  and  small  particles. 


RELATION  OF  BURSARIA  TO  FOOD 


7 


Figure  1,  II,  is  a sagittal  section  through  the  body  in  the  plane 
indicated  by  the  straight  line  through  figure  1,  /;  it  shows  the 
path  of  total  rejection,  arrows  A,  the  path  of  entrance  (by  heavy 
arrows  D)  and  the  path  of  rejection  of  smaller  particles  (by 
arrows  C). 

At  the  point  of  entrance  into  the  endoplasm  the  transport  of 
the  accepted  particles  is  brought  about  not  alone  by  the  cilia 
but  also  if  not  exclusively,  in  the  case  of  larger  particles,  by  a 
peristaltic  wave  in  the  wall  of  the  oral  cavity  behind  the  par- 
ticle pushing  it  into  the  body. 

FORMATION  OF  THE  VACUOLE  AND  THE  ELIMINATION  OF  RESIDUES 

The  vacuoles  when  formed  always  contain  some  liquid,  though 
at  times  the  amount  may  be  very  small.  The  size  and  shape 
of  the  vacuoles  varies  greatly  and  depends  upon  the  kind  of 
food  eaten,  and  upon  many  other  conditions,  as  will  be  shown. 
Often  the  food  forms  large  irregular  masses,  which  in  the  case  of 
fresh  yolk  may  so  completely  fill  the  body  that  after  a half- 
hour  or  more  of  feeding  the  dorsal  side  of  the  body  cortex  is 
burst  open  and  the  food  mass  is  extruded.  The  opening  then 
closes  and  the  organism  again  assumes  its  usual  shape.  The 
rate  of  formation  of  the  vacuoles  is  intimately  bound  up  with 
the  same  complex  conditions  which  determine  their  size  and 
shape.  The  circulation  of  the  vacuoles  in  Bursaria  is  not  re- 
ducible to  any  definite  order,  such  as  has  been  shown  to  exist 
more  or  less  definitely  in  Paramecium,  by  Metalnikow  (T2)  and 
others,  and  in  Carchesium  by  Greenwood  (^94).  The  vacuoles 
often  become  lodged  in  one  place  and  there  digestion  is  com- 
pleted. This  may  often  be  seen  in  cases  where  fat-extracted 
yolk  particles  become  lodged  in  the  tail.  During  digestion  and 
resorption  the  large  vacuoles  usually  become  smaller  and  any 
residual  contents  are  finally  extruded.  The  residues  are  always 
extruded  from  a small  area  on  the  mid-dorsal  side  of  the  body 
of  the  organism.  This  may  readily  be  demonstrated  by  feeding 
the  animals  Chinese  ink.  The  changes  which  take  place  in  the 
food  vacuole  from  its  formation  to  its  disappearance  will  be 
considered  in  detail  in  a later  paper. 


8 


E.  J.  LUND 


MEASUREMENT  OF  THE  AMOUNT  OF  FOOD  EATEN  AND  METHOD 
OF  EXPERIxMENTATION 

In  order  to  express  quantitatively  the  relations  of  Bursaria 
to  food,  it  is  necessary  to  obtain  a reliable  method  for  measuring 
the  amount  of  food  taken  in  a given  length  of  time,  under  given 
conditions.  The  unit  of  volume  employed  was  that  of  one 
grain  of  fresh  hard-boiled  yolk  of  hen’s  egg.  The  eggs  were 
boiled  fifteen  minutes.  These  grains  are  readily  eaten  by  Bur- 
saria and  may  be  obtained  of  an  approximately  uniform  size. 
It  is  necessary  to  deal  with  suspensions  of  such  grains,  having 
a uniform  concentration  (that  is,  containing  the  same  number 
of  grains  to  a given  volume).  For  this  purpose  stock  suspen- 
sions were  made  up  on  successive  days  from  yolk  of  the  same 
egg:  these  were  made  uniform  by  making  them  up  in  vials  of 
the  same  size  and  comparing  each  with  a standard  concentration 
kept  in  a vial  of  the  same  size.  Various  known  grades  of  con- 
centration were  then  made  up  by  adding  a known  volume  of 
the  stock  suspension  to  5 cc.  of  water  in  a stender  dish  of  8 cc. 
capacity.  This  procedure  was  found  to  be  sufficiently  accurate 
to  avoid  the  introduction  of  any  observable  variation  in  the 
amount  of  yolk  eaten  in  a measured  period  of  time  (page  20. 2) 
Uniformity  in  the  size  of  the  yolk  grains  was  obtained  by  repeat- 
edly washing  the  fresh  hard  boiled  yolk  crystals  in  distilled  or 
tap  water  and  decanting,  until  the  suspension  when  left  to  settle 
leaves  a clear  supernatant  liquid.  The  smaller  grains  remain 
in  suspension  a little  longer  than  the  larger  ones  and  thus  may 
be  removed  by  decantation.  Uniformity  in  size  is  still  further 
obtained  by  drawing  off  the  grains  from  the  same  level  in  the 
clear  suspension  with  a pipette.  Some  eggs  have  yolk  crystals 
of  more  uniform  size  than  others,  so  that  only  the  eggs  best  in 
this  respect  have  been  used. 


2 In  most  of  the  experiments  it  was  necessary  to  make  up  only  a single  stock 
suspension,  since  the  animals  were  fed  only  once  and  all  the  feeding  was  carried 
out  at  the  same  time.  In  the  case  of  experiments  which  required  the  feeding  of 
yolk  on  more  than  one  day,  however,  this  standard  concentration  had  likewise 
to  be  made  up  anew  each  day  by  comparison  with  that  of  the  day  before. 


RELATION  OF  BURS  ARIA  TO  FOOD 


9 


The  uniformity  in  size  of  the  yolk  grains  is  of  course  of  para- 
mount importance  in  many  of  the  experiments  and  for  some  of 
the  conclusions  which  will  be  drawn  from  them.  In  order  that 
the  degree  of  uniformity  might  be  tested  and  indicated  quanti- 
tatively, a large  number  of  measurements  of  grains  of  the  pre- 
pared yolk  suspension  were  made  at  different  times  by  means 
of  a stage  micrometer.  The  following  shows  a typical  result 
of  one  set  of  these  measurements:  105  grains  were  measured  and 
the  numbers  divided  at  random  into  three  sets  of  35  each  and 
the  average  of  the  diameters  taken.  These  gave  respectively 
0.0890,  0.0906,  and  0.0837  mm.  The  range  of  variation  of  the 
diameter  was  from  0.060  to  0.130  mm.  The  distribution  of  the 
variations  are  shown  by  the  following  figures: 


Diameter  of  grain  mm. 0.06  0.07  0.08  0.09  0.10  0.11  0.12  0.13 

Frequency 12  19  16  14  20  13  6 5 


By  thus  being  able  to  obtain  a very  constant  average  diameter 
of  a comparatively  small  number  (30  to  50  grains)  the  errors 
introduced  by  the  individual  variation  in  size,  which  in  the  above 
example  is  about  as  § is  to  1,  is  largely  eliminated.  In  order  to 
remove  the  objection  to  experimental  results  based  on  the  vol- 
ume of  granules  of  varying  size,  a large  number  of  individuals 
(20  to  100,  depending  upon  the  purpose  of  the  experiment)  were 
used  in  each  experiment  and  the  number  of  grains  eaten  was 
counted;  furthermore  the  experiments  were  always  repeated 
whenever  there  could  be  any  doubt  as  to  the  validity  or  signifi- 
cance of  the  results.  Hence,  as  will  be  shown  later,  the  lack  of 
strict  individual  uniformity  of  the  unit  volume  is  corrected  (a) 
by  the  fact  that  the  average  size  of  the  yolk  grain  is  practically 
constant,  (b)  by  using  a large  number  of  individuals  in  each 
experiment,  and  (c)  by  repeating  the  experiment. 

Thus  having  the  form,  weight  and  volume  of  the  units  of  food 
eaten  made  practically  constant,  we  may  vary  one  of  their  prop- 
erties— as  for  example,  their  chemical  nature — by  letting  them 
adsorb  different  kinds  of  toxic  and  non-toxic  substances  which 
are  diffusible  or  non-diffusible  in  the  native  medium,  tap  or  dis- 
tilled water.  We  may  therefore  test  the  responses  to  variations 


E.  J.  LUND 


• 10 

in  this  one  property — namely,  the  chemical  nature  of  the  grain 
— and  its  effects. 

An  approximately  constant  medium  was  provided  by  using 
tap  water.  This  precaution  is  important,  for,  as  will  be  shown, 
the  nature  of  the  medium  often  affects  or  determines  the  kind 
of  results  which  are  obtained.  Distilled  water  was  also  used 
but  it  was  found  that  this  extra  precaution  was  not  necessary 
in  most  of  the  experiments,  and  since  distilled  water  is  toxic 
if  the  organisms  are  left  in  it  too  long  or  the  change  is  too  rapid, 
it  could  not  have  been  used  in  many  of  the  experiments,  even 
if  it  had  been  otherwise  desirable  to  do  so. 

The  organisms  were  starved  in  400  cc.  of  tap  water  for  eight- 
een to  twenty-four  hours  previous  to  each  experiment.  At  the 
end  of  this  time  they  were  free  from  food  and  residues.  Thus 
an  optically  clear,  active  and  perfectly  normal  cell  was  obtained 
with  which  to  begin  work  in  all  the  experiments  where  uniform- 
ity in  this  respect  was  desired.  All  the  factors  with  which  we 
are  dealing  except  the  ^physiological  states’  of  the  organisms 
themselves  are  known  and  uniform  to  within  narrow  limits,  while 
the  one  of  which  we  wish  to  test  the  effects  can  be  controlled 
and  varied. 

INTERNAL  RELATIONS  AFFECTING  THE  FEEDING  PROCESS 

1.  The  relation  of  the  physiological  state  of  the  organism  to  the 

feeding  process 

By  the  words  ^physiological  state’  is  here  meant  the  condition 
as  a whole,  of  the  equilibria  in  the  physical  and  chemical  reac- 
tion system,  the  cell,  at  a certain  time  in  the  duration  of  its 
existence.^ 

This  condition  or  state  is  to  be  thought  of  as  being  limited  to 
the  space  which  the  organism  occupies,  or  is,  in  other  words, 
internal.  However,  it  is  obviously  absurd  for  anyone  to  attempt 

3 This  definition  is  justified  because  in  so  far  as  the  facts  are  at  present 
known,  this  is  the  only  kind  of  system  with  which  we  have  to  deal  in  the  cell, 
and  therefore  in  the  present  state  of  knowledge  the  only  logical  universal  assump- 
tion for  experimental  purposes  is  to  define  ‘physiological  states’  in  terms  of  such 
known  systems,  until  the  universality  of  the  assumption  is  disproved. 


RELATION  OF  BURSARIA  TO  FOOD 


11 


a definite  and  strict  separation  of  the  internal  and  external  of 
any  living  organism,  and  especially  is  this  true  of  the  cell.  Yet 
for  purposes  of  presentation,  this  becomes  highly  convenient, 
and  it  is  only  for  this  purpose  that  the  above  rough  distinction 
is  made  here.  When  all  external  conditions  'are  made  the  same  in 
two  experiments  which  nevertheless  give  different  results,  the  differ- 
ences must  he  attributed  to  different  conditions  within  the  organism, 
and  it  is,  as  a rule,  only  in  this  way  that  different  physiological 
states  are  at  present  practically  perceptible. 

Differences  in  physiological  state  in  unicellular  animals  are 
made  evident  most  readily  in  the  relations  to  food,  as  may  be 
seen  from  the  work  of  Metalnikow  (’12)  on  Paramecium  and  by 
Schaeffer  (’10)  on  Stentor. 

Bursaria  affords  most  excellent  material  for  the  elucidation  of 
the  relation  of  these  dynamic  states  to  the  feeding  process  and 
of  the  fact  that  this  relation  changes  while  the  external  con- 
ditions remain  constant.  These  facts  are  brought  out  in  the 
following  experiments  by  using  both  single  individuals,  and  large 
numbers  of  individuals  collectively,  at  the  same  time,  and  analyz- 
ing the  results. 

The  total  quantity  eaten  and  the  rate  of  feeding.  Table  1 
gives  the  results  of  a typical  experiment  designed  to  show  the 
difference  in  the  total  quantity  of  food  eaten  and  also  the  differ- 
ence in  the  rate  of  feeding  of  Bursaria  from  different  cultures. 

Material  from  two  different  cultures,  A and  B,  was  starved 
twenty-four  hours  in  each  of  two  dishes  containing  400  cc.  of 
tap  water.  1 cc.  of  a fresh  hard  boiled  yolk  suspension  was  placed 
in  each  of  16  stender  dishes  of  8 cc.  capacity;  5 cc.  of  tap  water 
was  then  added  to  each.  Thirty  individuals  from  culture  A 
were  placed  in  each  of  8 of  the  dishes.  Alternately  with  these 
8 sets  from  culture  A were  placed  8 sets  of  thirty  individuals  each 
from  culture  B in  the  other  8 dishes.  At  the  end  of  the  time 
intervals  noted  in  the  table,  in  each  case,  the  contents  (6.5  cc.) 
of  one  dish  each  of  A and  of  B were  transferred  into  a stender 
dish  with  500  cc.  of  tap  water.  This  stops  the  feeding.  The 
individuals  were  then  immediately  picked  out  of  these  large 
dishes,  placed  in  8 cc.  dishes  and  killed  in  Aleves’  fluid.  The 


12 


E.  J.  LUND 


counts  of  the  number  of  grains  contained  in  each  individual  were 
taken  at  the  end  of  the  experiment. 

Table  1 shows  (1)  that  Bursariae  living  in  the  two  different 
cultures  differ  in  the  total  amount  of  food  eaten  in  the  same 
length  of  time.  In  other  cases,  of  course,  individuals  from  di- 
verse cultures  will  give  identical  results  so  far  as  feeding  is  con- 
cerned, while  two  or  more  different  cultures  may  also  differ  to 
a greater  extent  than  the  above  table  shows.  Moreover,  the 
amount  of  food  eaten  by  a gi^'en  culture  may  vary  at  different 
times.  The  greater  the  length  of  time  of  feeding  (within  certain 
limits)  the  greater  the  total  amount  of  food  eaten.  Not  only 
does  the  total  amount  of  food  taken  differ  in  the  two  cultures, 
but  what  is  equally  important,  (2)  the  rate  of  feeding  varies  with 
organisms  from  different  cultures.  This  was  observed  in  numer- 
ous other  experiments.  Under  some  conditions  the  animals  fill 
their  bodies  quickly,  while  at  other  times  this  takes  place  slowly; 
or  only  a small  number  of  grains  may  be  eaten. 

The  facts  are  shown  most  clearly  by  the  curves  A and  B, 
figure  2,  representing  the  number  of  grains  of  yolk  (ordinates) 
eaten  by  the  thirty  individuals  in  successive  periods  of  one-half 
minute  (abscissae)  throughout  the  time  of  the  feeding  process. 
Curve  A is  plotted  from  the  results  of  culture  A and  curve 
B from  those  of  culture  B^  in  table  1.  The  immediate  rapid 
rise  of  curve  A shows  that  the  rate  of  feeding  of  culture  A dur- 
ing the  first  six  successive  periods  of  one-half  minute  each  was 
about  from  five  to  twenty  times  as  great  as  in  any  of  the  subse- 
quent fifty-seven  minute  intervals.  A similar  high  initial  rate 
is  also  shown  by  curve  B (culture  B),  but  here  the  rise  to  the 
maximum  was  not  so  steep  and  the  rate  during  the  first  six  half- 
minute periods  was  only  about  from  four  to  ten  times  the  rate 
during  the  subsequent  fifty-seven  half-minute ‘intervals. 

In  order  to  show  more  clearly  that  the  results  apply  to  the 
individuals  taken  separately  as  well  as  to  the  averages  for  all 
(i.  e.,  to  the  cultures  as  a whole)  the  data  may  be  arranged  as 
in  table  2.  As  this  table  shows,  at  the  end  of  sixty  minutes 
all  but  an  insignificant  number  of  animals  from  each  culture 
had  eaten  yolk  grains:  hence,  the  difference  in  the  amount  and 


RELATION  OF  BURSARIA  TO  FOOD 


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14 


E.  J.  LUND 


rate  of  food  taken  by  the  two  cultures  was  not  due  to  some 
sporadic  difference  caused,  for  example,  by  a very  high  rate  of 
feeding  by  a few  individuals  and  no  food  eaten  by  others,  but 
rather  to  a uniform  difference  between  the  sets  of  individuals 
from  the  two  cultures.  Therefore  the  results  are  typical  for 
the  individual  as  well  as  for  the  culture  as  a whole.  Moreover, 
if  we  calculated  the  averages  of  A and  B on  the  basis  of  those 
individuals  alone  which  had  one  or  more  grains,  the  average  of 
A would  still  be  greatly  in  excess  of  that  of  B. 


Fig.  2 Showing  the  rates  of  feeding  by  the  two  cultures  A and  B,  curves  A 
and  B,  respectively.  Plotted  from  the  results  of  table  1. 

We  may  express  the  variation  in  the  total  quantity  eaten  by 
the  standard  deviation  of  each  corresponding  group  of  thirty 
individuals  in  A and  B,  as  is  done  in  the  last  column  of  table  2. 
The  reciprocal  of  the  standard  deviation  (o-)  is  a measure  of  the 
degree  of  uniformity  among  the  individuals.  It  will  be  noted 
that  there  is  an  increase  in  the  range  of  variation  and  the  stand- 
ard deviation  with  increase  in  the  length  of  time  of  feeding;  this 
means  that  the  difference  in  physiological  state  among  indi- 
\dduals  of  the  same  culture  finds  a fuller  and  more  definite  ex- 


TABLE  2 

Showing  the  differences  in  'physiological  state  of  Bursaria  from  two  Cultures,  A and  B.  The  feeding  process  is  used  as  an  index 


RELATION  OF  BURSARIA  TO  FOOD 


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16 


E.  J.  LUND 


pression  in  the  results  of  the  experiment  as  the  length  of  time 
of  feeding  is  increased.  The  final  total  number  of  grains  eaten 
when  the  time  is  long  is  then  a more  accurate  index  of  the  rela- 
tion of  the  physiological  state  to  the  feeding  process  than  if  the 
time  of  feeding  is  short.  The  greater  this  difference  in  the  total 
quantity  of  food  eaten  the  greater  is  the  difference  in  the  physio- 
logical state  of  the  different  individuals.  We,  therefore,  have 
in  the  amount  of  food  eaten,  if  the  length  of  time  of  feeding 
is  long  enough,  a fairly  good  relative  measure  of  the  physiological 
state  of  the  single  individual  and  the  differences  in  the  physio- 
logical state  between  different  individuals  as  regards  their  rela- 
tion to  food  at  that  particular  time. 

2.  Changes  in  the  'physiological  state  as  shown  by  using  the  feeding 

process  as  an  index 

If  change  in  the  dynamic  conditions  of  the  cell,  as  regards  the 
food  relation,  does  occur,  this  should  be  observable  by  a change 
in  the  feeding  process,  and  such  is  indeed  the  fact.  This  is 
shown  in  table  3.  Material  from  cultures  C and  D was  starved 
in  tap  water  for  twenty-four  hours.  Five  active  individuals 
were  then  picked  out  from  each  and  tested  individually.  Thej^ 
were  fed  twenty  minutes  and  each  one  was  observed  continuously 
during  the  experiment.  The  number  of  grains  eaten  and  re- 
jected and  the  time  as  called  off  by  the  observer  were  noted.'* 
In  this  way  the  time  record  of  the  relation  of  acceptance  and 
rejection  of  food  was  obtained.  The  yolk  concentration,  tem- 
perature, and  so  forth  were  the  same  in  all  the  tests. 

As  the  table  shows,  yolk  grains  were  at  first  rapidly  eaten. 
At  the  end  of  the  first  few  one-half  minute  intervals  the  action 
of  the  cilia  was  frequently  reversed,  thus  rejecting  the  food  after 
it  had  been  taken  into  the  oral  apparatus.  There  was,  therefore, 
a definite  change  from  eating  to  the  rejection  of  food  by  the 
feeding  mechanism.  This  change  was  more  rapid  in  general, 
in  the  individuals  from  culture  C than  in  those  from  culture  D. 

^ I am  indebted  to  Mr.  K.  S.  Lashley  for  kindly  aiding  me  in  taking  the  records 
of  this  experiment. 


TABLE  3 

ii'pril  4'-  Culture  C 

Starved,  tap,  24  Iwjurs  Tests  on  individuals 


RELATION  OF  BURSARIA  TO  FOOD  17 


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f Accepted . . 

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THE  JOURNAL  OF  EXPERIMENTAL  ZOSLOGY,  VOL. 


16,  NO.  1 


18 


E.  J.  LUND 


These  results  from  individuals  are  therefore  strictly  comparable 
and  in  accord  with  those  obtained  when  a large  number  are 
tested  at  one  time  (table  1). 

Now,  in  order  to  explain  the  cause  of  the  change  in  reaction 
the  suggestion  might  be  offered  that  Bursaria  shows  a decrease 
in  the  rate  of  feeding  because  of  the  decrease  in  the  amount  of 
space  in  the  body  which  food  can  occupy.  This  is  undoubtedly 
true  to  some  extent  in  those  individuals  which  do  not  stop  feed- 
ing until  the  cell  becomes  distorted  by  the  comparatively  immense 
mass  of  food.  So  far  as  the  volume  capacity  of  a normal  indi- 
vidual of  Bursaria  is  concerned,  hundreds  of  observations  have 
shown  me  beyond  doubt  that  this  may  frequently  be  as  much 
as  twenty-five  to  thirty  grains.  Nevertheless,  reversal  of  the  cilia 
always  takes  place  sooner  or  later.  But  the  suggestion  evidently 
does  not  apply  to  those  individuals  which  show  a change  in  the 
reaction  when  only  a few  grains  have  been  eaten,  for  it  seems 
impossible  to  understand  how  there  could  be  a difference  of  as 
much  as  twenty  grains  of  fresh  yolk  (table  2)  in  two  normal 
individuals  of  equal  size,  when  the  cells  are  under  exactly  the 
same  conditions,  if  this  result  were  not  due  to  a difference  in  the 
physiological  state  of  the  cells.  Change  in  feeding  was  caused 
by  the  periodic  reversal  of  the  cilia  and  the  reversal  of  the  cilia 
in  turn  in  some  manner  initiated  or  caused  by  a stimulus  from 
the  food  already  eaten,  for  it  seems  most  natural  to  suppose 
that  the  stimulus  originated  from  the  change  produced  by  the 
food  mass  in  the  interior  of  the  cytoplasm.  The  most  definite 
evidence  that  the  change  is  due  to  stimulus  from  the  eaten  food 
is  found  in  the  radical  change  in  the  action  of  the  cilia  of  the 
feeding  mechanism. 

If  such  fed  individuals  as  those  in  table  3 are  left  in  tap  water 
free  from  food  they  may  again  eat  yolk  after  digestion  is  ‘par- 
tially or  wholly  completed,  and  again  show  a similar  decrease 
in  the  rate  of  feeding,  that  is,  a reversal  of  the  oral  cilia.  The 
total  quantity  which  will  be  eaten  may  be  greater  than  that 
eaten  at  the  previous  feeding;  but  it  usually  is  less,  or  often  none 
at  all. 


RELATION  OF  BURSARIA  TO  FOOD 


19 


The  process  of  feeding  in  Bursaria  shows  it  to  be  a function- 
ally equilibrating  system  in  its  behavior  towards  food  and  the 
condition  of  its  equilibrium  at  any  particular  time  constitutes 
the  physiological  state  which  the  cell  is  in,  so  far  as  its  relation 
to  food  is  concerned.  The  changes  in  the  increase  or  decrease 
in  the  quantity  of  food  eaten  in  successive  meals  and  the  increase 
or  decrease  in  the  rate  of  feeding  might  be  discussed  in  the 
psychological  terms  ‘hunger  and  ‘satiation/  but  it  is  evident 
that  the  simpler  terms  quantity  and  rate  express  the  facts  of 
experiment,  while  any  attempt  at  definitely  determining  whether 
the  changes  in  quantity  and  rate  are  the  same  or  different  from 
‘hunger’  and  ‘satiation’  will  obviously  lead  nowhere.  Hence  it 
seems  better  to  use  the  terms,  rate  and  quantity,  which  have  a 
clear  and  quantitative  meaning. 

3.  Other  causes  of  individual  variation 

Bursaria  at  times  closes  up  its  oral  apparatus.  This  may  take 
place  to  such  an  extent  that  the  opening  is  smaller  than  the 
food  particles  and  then  the  latter  can  of  course  not  be  eaten. 
This  condition  can  readily  be  observed  under  the  binocular  and 
it  can  always  be  determined  beforehand  whether  closure  has 
taken  place  to  such  an  extent  that  the  organisms  can  not  feed. 
Other  minor  accidental  individual  variations  are  also  present  to 
some  extent.  These  may  be  partly  due  to  the  difference  in  the 
size  of  the  grains  of  yolk  eaten.  Sometimes  when  an  individual 
is  weak,  owing  to  prolonged  starving  or  for  some  other  reason, 
two  or  three  grains  may  become  stuck  in  the  oral  pouch  and  this 
prevents  feeding  until  the  animal  succeeds  in  throwing  them 
out  or  by  other  means  they  become  loosened.  The  material 
used  was  always  examined  beforehand  to  make  sure  that  it  was 
in  a healthy  condition  so  that  these  accidental  conditions  play 
no  part  in  the  final  results  of  the  experiments  described. 

Such  a series  of  experiments  as  the  forego ’ng  do  not  show  us 
specifically  what  these  complex  conditions  are  which  have  been 
cloaked  in  the  phrase  ‘physiological  states.’  This  however  is 


20 


E.  J.  LUND 


not  the  object  of  the  above  experiments:  they  are  only  here 
considered  for  the  purpose  of  demonstrating  the  existence  of 
these  conditions,  the  fact  of  change  within  them  and  especially 
in  this  connection  their  role  in  the  external  phenomena  of  feeding 
and  food  selection  in  Bursaria,  and  how  they  may  affect  the 
results  which  will  be  given  in  the  following  pages. 

EXTERNAL  RELATIONS  OF  THE  FEEDING  PROCESS 
1,  Effects  of  external  factors  on  feeding 

a.  Concentration  of  the  food  supply.  The  rate  of  feeding  is 
within  comparatively  wide  limits  not  dependant  upon  the  con- 
centration of  the  yolk  suspension,  provided  it  is  not  too  low. 
This  may  be  illustrated  from  one  out  of  a series  of  experiments. 
The  time  of  feeding  was  reduced  to  five  minutes  for  the  purpose 
of  bringing  out  the  effect  of  difference  in  the  concentration  more 
strongly  If  the  animals  had  been  left  in  the  suspensions  twenty 
minutes  (the  usual  time  of  feeding;  cf.  table  3)  the  difference 
would  have  been  less  marked,  especially  with  rhaterial  which 
shows  a high  rate  of  feeding. 

Experiment  Material  from  a healthy  culture  was  starved  twenty- 
four  hours  in  tap  water.  All  were  perfectly  normal  and  active.  The 
experiment  was  carried  out  in  8 cc.  stender  dishes.  The  concentration 
in  dish  B was  8 times  that  in  dish  A.  Twenty  individuals  were  placed 
in  each  dish.  The  results  from  trial  number  2 represent  more  nearly 
the  ideal  because  these  two  suspensions  were  kept  uniformly  distrib- 
uted during  the  five  minutes  feeding,  and  the  individuals  were  picked 
out  alternately  by  fives.  Both  trials,  however,  express  equall}^  well 
the  proportional  effect  of  concentration,  namely,  1 to  2,  as  compared 
to  the  proportion  of  concentration,  1 to  8®  (table  4). 

The  concentrations  used  in  the  experiment  are  approximately 
represented  by  figure  3. 

® The  experiments  given  in  this  paper  are  numbered  in  regular  order  for  the 
convenience  of  the  reader,  and  do  not  represent  the  actual  order.  Only  a small 
number  of  the  experiments  actually  carried  out  are  given. 

® In  all  the  experiments  considered  in  this  paper,  where  the  concentration 
plays  a part,  the  concentration  was  intermediate  between  those  used  in  this 
experiment  (fig.  3). 


RELATION  OF  BURSARIA  TO  FOOD 


21 


TABLE  4 
Experiment  I 


2 

4|  2 

1 

1 

2 

1 

5 5 

3 

0 

1 

4j0 

4 3 

0 

4 1 

2 

45 

2.25 

1 

8 6 

1 

4 

4 

3 

81  4 

1 

5 

7 

2;  4 

1 

0|7 

2 

81 

4.05 

Fig.  3 Showing  the  relative  concentration  of  yolk  in  dishes  A and  B of  Ex- 
periment 1. 


h.  Effect  of  mechanical  stimulation  and  of  mechanical  injury 
on  feeding. 

Experiment  II.  Thirty  individuals  from  the  same  culture,  starved 
twenty-four  hours,  were  placed  in  each  of  six  8 cc.  dishes  containing 
5 cc.  tap  water.  Before  feeding,  the  animals  in  three  of  the  dishes 
(Set  1 in  the  experiment)  were  mechanically  stimulated  by  means  of 
a pipette.  The  opening  of  the  latter  was  about  ten  times  the  width 
of  Bursaria.  The  edges  of  the  opening  were  made  smooth  by  melting. 
The  animals  of  Set  1 were  stimulated  by  drawing  them  up  into  the 
pipette  along  with  the  tap  water  in  the  8 cc.  dishes,  four  times.  Equal 
quantities  of  yolk  suspension  were  now  added  to  all  the  dishes.  After 
having  fed  ten  minutes  the  animals  of  Set  1 were  again  stimulated  by 
drawing  them  along  with  the  yolk  suspension  into  the  pipette  two 
times;  at  the  same  time  the  control.  Set  2,  was  stirred  by  gently  shak- 
ing the  dish  and  not  allowing  any  instrument  to  touch  the  animals; 
hence  the  distribution  of  the  yolk  was  the  same  in  the  two  sets  of 
dishes.  All  the  individuals  in  Set  1,  after  having  been  stimulated, 
were  perfectly  normal  and  not  injured.  They  looked  like  those  of  the 


22 


E.  J.  LUND 


TABLE  5 
Experiment  II 
Set  1.  Stimulated 


DISH 

NUMBER 

; OF 

GRAINS 

EATEN  BY 

EACH  INDIVIDUAL 

TOTAL 

AVG.  PER 

IND. 

A 

0 

1 

4 

0 

1 

0 

1 

3 

! 

1 

5 

1 

1 

2 

0 

1 

1 

0 

1 

! 

1 1 

0 

2 

2 

0 

1 

3 

0 

0 

0 

0 

33 

grains 

1.1 

B 

1 

0 

0 

2 

0 

0 

! 0 

0 

2 

0 

2 

0 

0 

0 

2 

0 

0 

3 

0 0 

1 

0 

1 

1 

0 

0 

0 

0 

1 

1 

17 

0.56 

C 

4 

0 

1 

2 

1 

6 

0 

i' 

1 

1 

3 

3 

1 

1 

3 

0 

0 

4 

0 

3 

0 

0 

3 

3 

48 

1.60 

Total  average. . 

1.08 

Set  2:  Control;  Not  Stimulated 


DISH  NUMBER  OF  GRAINS  EATEN  BY  EACH  INDIVIDUAL  TOTAL 


1 

grains 

A 

3 

5 

7 

2 

3 

6 3 

3 

3 

4 

14 

13 

3 

1 

2 

5 

8 

2 

9 

5 

9 

5 

10 

3 

7 

7 

6 

3 

4 

2 

157 

5.23 

B 

2 

1 

3 

6 

3 

1 5 

1 

3 

6 

4 

4 

5 

2 

3 

2 

5! 

6 

11 

4 

2 

6 

6 

1 

3 

2 

7 

2 

3 

0 

109 

3.63 

C 

6 

1 

i 

3 

6 

5 

e'  5 

3 

2 

7 

4 

4 

4 

3 

' 3 

6 

5 

4 

3' 

2 

6 

1 

3 

2 

3 

5 

6 

2 

2 

li 

113 

3.76 

! 1 

1 

1 i 

1 

Total  average 4 . 206 


control.  If  a smaller  pipette  is  used  or  a larger  one,  and  the  stimu- 
lation, by  sucking  them  along  with  the  medium  up  into  the  pipette, 
is  more  violent,  it  will  stimulate  and  injure  the  organisms  so  that  they 
will  not  eat  at  all,  or  at  least,  not  for  some  time  after  stimulation.  Of 
course  structural  injuries  are  very  easily  produced,  with  the  result  that 
regulation  of  the  cell  must  take  place  before  any  food  can  be  eaten 
(table  5). 

Proof  that  in  this  experiment,  Set  1,  if  not  in  Set  1 of  Experi- 
ment III,  the  organisms  were  not  injured  beyond  the  capacity 
for  swallowing,  is  found  in  the  fact  that  the  great  majority  did 
eat,  though  only  a comparatively  small  number  of  grains  An- 
other experiment  may  be  given  to  illustrate  the  same  fact. 

Experiment  III.  The  animals  in  Set  1 were  not  stimulated  before 
feeding,  but  after  they  had  fed  for  five  minutes  they  were  stimulated 
by  drawing  the  suspension  with  the  animals  in  it,  up  into  the  pipette 
only  once.  Material  from  a different  culture  was  used  in  this  experi- 
ment; time  of  feeding  fifteen  minutes.  The  control  suspension  with 
the  organisms  was  redistributed  once  by  gently  shaking  the  dish.  The 
animals  were  all  normal  in  form  at  the  end  of  the  experiment  (table  6). 

In  Experiment  III  the  stimulus  was  only  slight  as  compared 
to  that  in  Experiment  II,  yet  the  effect  was  marked  As  stated 
above,  strong  stimulation  may  totally  prevent  feeding. 


RELATION  OF  BURSARIA  TO  FOOD 


23 


TABLE  6 
Experiment  III 
Set  1 : Stimulated 


NUMBER  OF  GRAINS  EATEN  BY  EACH  INDIVIDUAL 


A 

5 

4 

1 

6 

8 

6 

9 

1 

Oi  7 

0 

10 

3 

0 

1 

3 

3 

4 

0 

1 

4 

6 

5 

1 

5 

0 

0 

0 

4 

0 

0 

96 

grains 

3.2 

B 

0 

0 

4 

0 

4 

15 

11 

Oj  0 

9 

0 

1 

4 

6 

5 

2 

5 

8 

4 

0 

9 

6 

6 

1 

14 

5 

6 

0 

2 

0 

127 

4.0 

C 

5 

3 

0 

4 

0 

2 

1 

3 5 

0 

3 

1 

0 

1 

0 

0 

4 

2 

0 

3 

1 

3 

3 

i 

0 

8 

0 

0 

3 

L_ 

0 

4 

59 

1.96 

•AVG.  PER 
IND. 


Total  average. 


3.5 


Set  2;  Control:  Not  Stimulated 


A 

B 

C 


NUMBER  OF  GRAINS  EATEN  BY  EACH  INDIVIDUAL 


1 

1 

I 

grains 

6 

9 

8 

6 

6|  4 

10 

71  6 

5 

9 

7 

7 

1 

7 

2 

5 

9 

9 

14 

6 

7 

6 

7 

6 

0 

10 

7 

9' 

6 

201 

6.70 

6 

2 

4 

6 

*1  ' 

7 

9|  5 

4 

6 

4 

6 

2 

7 

1 

10 

0 

4 

7 

2 

5 

5 

5 

6 

7 

10 

3 

152 

5.06 

0 

11 

4 

I 

6 

69 

8 

6 6 

9 

6 

1 

5 

1 

5 

9 

7 

7 

9; 

2 

7 

4 

7 

8 

8 

7 

12 

6 

8 

6 

9 

1 

198 

6.60 

1 

AVG.  per 
IND. 


Total  average 


6.12 


The  effect  of  mechanical  stimulation  must  be  emphasized  be- 
cause it  shows  that  in  any  work  of  this  nature  it  is  necessary  to 
handle  the  organisms  gently.  This  relation  must  be  inferred  to 
apply  to  work  on  other  Infusoria  also,  at  least  to  some  extent, 
c.  Effect  of  temperature  on  feeding. 

Experiment  IV.  Thirty  individuals  starved  twenty-four  hours,  were 
placed  in  each  of  six  vials.  Each  vial  contained  5 cc.  of  tap  water. 
These  vials  were  now  placed  in  large  dishes  containing  water  kept  at 
the  desired  temperatures.  The  latter  were  read  on  a small  thermometer 
set  inside  of  each  vial.  Equal  quantities  of  fresh  yolk  suspension  were 
added  when  the  temperature  had  reached  the  desired  point.  They 
were  fed  fifteen  minutes  (table  7). 

TABLE  7 
Experiment  IV 


NUMBER  OF  GR.AINS  EATEN  BY  EACH  INDIVIDUAL  TOTAL 


deg.  C. 

5 

0 

0 

0 

0 

0 

0 

0 

0 0 

0 

0 

0 0 

0 

0 

Oj  0 

0 

0 

0 

0 0 

0 

0 

0 

0 

0 

0 

0 

1 

0 

1 

0 

10 

0 

0 

0 

0 

0 

0 

2 

oi  0 

0 

0 

0 0 

0 

0 

0 0 

0 

0 

0 

0 0 

1 

0 

0 

0 

0 

0 

0 

0 

3 

15 

i 2 

0 

1 

3 

1 

1 1 

3 

4 2 

1 

0 

0 0 

6 

4 

0 3 

5 

1 

0 

2 3 

1 

0 

1 

0 

0 

4 

0 

4 

52 

20 

9 

12 

4 

19 

6 

9 

8 

2 0 

6 

2 

13I14 

6 

5 

9|  2 

Q 

4 

2 

4 5 

4 

8 

7 

9 

6 

7 

5 

8 

195 

35 

1 0 

! 

10 

I 

3 

4 

1 

11 

1 

' 6 

9 

I3I  6 

1513 

1 i 

16  4I 

i 1 1 

612 

1 1 

0 9 

i 

15 

5 

i 

4 

8 13 

1 

91 

13 

4 

2 

4 

1 i 

8 

1 

10 

t 1 

9 

, 1 

241 

39]  i 

to  [■ All  died  in  from  5 to  10  minutes 

40j 


24 


E.  J.  LUND 


The  experiment  was  repeated  with  closely  similar  results.  At 
lower  temperatures  the  animals  are  always  unable  to  eat.  As 
the  temperature  is  raised  and  the  activity  of  the  cell  increases, 
the  rate  of  feeding  increases,  continuing  to  increase  nearly  up 
to  the  point  where  the  cell  is  injured  or  killed  by  the  heat.  At 
temperatures  between  20°  and  25°C.  (i.  \,  at  about  the  optimum) 
the  increase  in  the  rate  of  feeding  can  be  determined  only  by 
using  a very  large  number  of  individuals,  since  the  variations 
obliterate  the  effects  when  a small  number  is  used. 

All  the  experiments  relating  to  other  conditions  were  carried 
on  at  temperatures  ranging  between  20°  and  27°C.  Where  neces- 
sary (as  in  prolonged  experiments  on  digestion)  the  temperature 
was  kept  constant  to  within  1°  to  1.5°C.,  throughout  the  course 
of  the  experiment,  by  keeping  the  organisms  in  moist  chambers 
in  a constant  temperature  oven. 

d.  Effect  of  HCl  and  NaOH  on  the  feeding  reaction. 

Experiment  V.  The  medium  used  in  this  experiment  (table  8)  was 
conductivity  water. ^ Any  water  less  carefully  purified  is  worthless  for 
such  experiments,  as  was  shown  by  experiments  carried  out  with  tap 
water.  By  comparing  the  results  it  was  strikingly  evident  that  the 
acid  and  base  had  reacted  with  the  salts  and  other  impurities  in  the 
tap  water  and  hence  their  effect  was  removed  in  low  concentrations. 
The  animals  were  washed  once  in  conductivity  water  before  putting 
them  into  the  solutions.  Time  of  feeding,  twenty  minutes  (table  8). 

It  is  seen  from  table  8 that  the  base  NaOH  was  much  more 
toxic  than  the  HCl,  and  that  as  the  concentraton  was  increased 
the  number  of  grains  eaten  became  less  and  less.  The  chemical 
relations  of  the  food  and  medium  will  be  considered  in  more 
detail,  later  on  (p.  29). 

e.  Effect  of  strong  white  light  on  the  feeding  reaction.  Bursaria, 
when  kept  in  dishes  with  a rather  clear  medium,  often  collect 
in  the  greatest  number  on  the  side  of  the  dish  away  from  fairly 
strong  white  light.  It  therefore  became  of  interest  to  test  what 
effect  continuous  light  of  a high  intensity  would  have  upon  the 
rate  of  feeding. 

^ Prepared  and  used  in  the  Department  of  Physical  Chemistry  for  conductivity 
measurements. 


RELATION  OF  BURSARIA  TO  FOOD 


25 


TABLE  8 
Experiment  V 

NaOH 


1/400., 

1/600., 

1/800. 

1/1200. 


NUMBER  OF  GRAINS  EATEN  BY  EACH  INDIVIDUAL 

All  dead  in  3 minutes 

All  dead  in  10  minutes 

Many  dead  at  15  minutes;  none  eaten  at  end 

of  20  minutes 

All  alive  and  normal  in  shape  at  end  of  ex- 
periment; no  grains  of  yolk  eaten 


1/1600 

0 

1 

0 

0 

0 

1 

1 

0 

2 

1 

2 

2 

0 1 

3 

2 

0 3; 

0 

1/3200 

6 

8 

8 

7 

3 

5 

1 

3 

1 

9 

5 

5 

lo'  9 

6 

11 

4 7 

5 

1/6400 

5: 

9 

8 

8 

6 

14 

1 

8 

1 

11 

5 

8 

5 

1%  4 

1 

8 

11 

6 8 

1 

6 

CONTROL  IN  CONDUCTIVITY  WATER 


ojio  9 


9 4 


lOjlO 


12 


5 617 

I I 


8 84 


TOTAL 

NUMBER 

GRAINS 


0 

0 

0 

0 

19 

121 

148 


155 


H Cl 


1/400 1 All  dead  in  5 minutes ! 0 


1/600 

' 0 

1 

2 

1 

1 

1 

1 

2 

0 

2 

1 

1 

4 

2 

o| 

1 

2 

0 

5 

4 

; 1 

2 

1 

1 32 

1/800 

1 

3 

4 

5 

13 

4 

5 

5 

2 

6 

0 

'5 

1 4l 

4 

3 

3 

7 

5 

1 5 

6 

; 90 

1/1200. . 

6 

8 

1 

8 

7 

8 

6 

0 

6 

3 

8 

i 3, 

1 

5 

4 

1 

1 

211 

1 

i 90 

1/1600 

6 

5 

8 

8 

7 

9 

3 

12 

3 

2 

5 

8 

14 

6 

9 

3 

5 

5 

5 

1 123 

1/3200 

8 

7: 

15 

5 

11 

13 

8 

9 

0 

2 

4 

1 

s! 

8 

2 

10 

5 

5 

6 

8 

1 130 

1/6400 

5 

1012 

10 

9 

7 

9 

3 

14 

6 

10 

5 

9 

9 

10 

10 

7 

9 

1 

5 

3 

162 

CONTROL 

, IN  CONDUCTIVITY 

WATER 

1 

1 

12; 

12 

1 

1 

13j 

Ojll 

u! 

5 

6 

2 

0 

9 

7. 

8 

211i 

1 1 

3111 

3 

4 

137 

Experiments  VI  and  VII.  White  light  from  the  arc  of  an  Edinger 
apparatus  was  focused  upon  the  stage  so  that  a spot  of  light  1^  inches 
in  diameter,  of  a very  high  intensity,  was  obtained.  The  light  was 
filtered  through  a layer  of  water  1.5  cm.  in  thickness.  An  8 cc.  stender 
dish  containing  thirty  normal  individuals  was  placed  in  the  spot  of 
light  and  the  usual  quantity  of  yolk  suspension  added.  A control  was 
kept  in  weak  diffuse  daylight.  The  animals  were  fed  twenty  minutes. 
The  following  results  show  that  continuous  action  of  intense  white 
light  on  the  animals  does  not  have  any  effect  upon  the  rate  of  feeding. 
Two  experiments  with  controls  are  given  (tables  9 and  10). 


26 


E.  J.  LUND 


• TABLE  9 

Experiment  VI 

Strong  white  light 


5i  6 4i  3!  6 010  Ol  5 4 4 


1!  2!  41  3'  3|  7'  31  6l  7 7 81  6!  7 4 


146 


TABLE  10 
Experiment  VII 


Strong  white  light 


NUMBER  OP  GRAINS  EATEN  BY  EACH  INDIVIDUAL 


6 9 


11! 


10 


11 


grams 

154 


Control:  Diffuse  daylight 


/.  Effect  of  the  electric  current.  Weak  induction  currents  have, 
within  a limited  time,  no  noticeable  effect  upon  the  feeding,  as 
is  shown  by  the  following  results  from  two  separate  experiments, 
VIII  and  IX.  The  total  number  of  grains  eaten  by  20  individ- 
uals in  each  of  two  8 cc.  dishes  is  given  in  each  experiment:® 

Experiment  VIII 

Dish  A — 73,  total  number  of  grains  eaten  by  20  individuals 

Dish  B — 69,  total  number  of  grains  eaten  by  20  individuals 

Experiment  IX 

Dish  A — 65,  total  number  of  grains  eaten  by  20  individuals 

Dish  B — 64,  total  number  of  grains  eaten  by  20  individuals 

Control  for  Experiments  VIII  and  IX;  not  stimulated  by  the 
current 

Dish  C — 89,  total  number  of  grains  eaten  by  20  individuals 

* The  apparatus  was  arranged  in  such  a way  as  completely  to  prevent  any 
effect  of  substances  liberated  at  the  electrodes,  by  inserting  the  electrodes  in  a 
physiological  normal  NaCl  solution  in  each  of  two  8 cc.  dishes  and  from  these 
the  circuit  was  closed  through  the  other  two  8 cc.  dishes  containing  the  animals, 
by  means,  of  small  H tube  connections  filled  with  tap  water  and  plugged  loosely 
with  a wad  of  cotton. 


RELATION  OF  BURSARIA  TO  FOOD 


27 


When,  however,  a direct  current  is  used  of  such  strength  that 
the  organisms  can  be  made  to  go  to  one  side  or  the  other  by 
reversal  of  the  current  the  effect  becomes  more  or  less  apparent. 
Feeding  can  not  be  prolonged  to  twenty  minutes  with  a strong 
direct  current,  for  the  organisms  are  easily  injured.  To  obviate 
this,  time  of  feeding  was  limited  to  five  minutes.  The  animals 
were  made  to  swim  from  one  side  to  the  other  by  frequent  rever- 
sals of  the  current.  In  Experiment  X,  they  were  stimulated  by 
frequent  reversal  of  the  direct  current  during  the  first  minute 
of  feeding  and  then  left  to  feed  four  minutes  more  without  stim- 
ulation. In  Experiment  XI  they  were  stimulated  in  the  same 
way  during  the  whole  period  of  feeding.  Time  of  feeding,  five 
minutes  (tables  11  and  12). 

TABLE  11 
Experiment  X 

Stimulated  by  direct  current,  1 minute 


Control:  Xo  current 


4 6 


10 


7 7 


8 5 


4|loi  8 


118 


TABLE  12 
Experiment  XI 

Stimulated  by  direct  current,  5 minutes 


B 


5 

5 

11 

0 

2 

18 

3 

Ijll 

on 

1310 

0 

5'  7 

0 

1 

1 

1 

I 

122 


As  the  results  show,  feeding  was  not  discontinued  under  these 
conditions  of  strong  stimulation  by  the  current,  though  the  or- 


28 


E.  J.  LUND 


ganisms  show  a somewhat  sjnaller  total  number  of  grains  eaten 
than  in  the  controls,  in  the  same  length  of  time.  The  difference 
is,  however,  too  small  to  have  a clear  significance. 

The  strength  of  current  may  be  increased  but  usually  feeding 
can  never  be  totally  inhibited  unless  the  organisms  are  injured 
or  killed  immediately  after  the  yolk  has  been  added. 

The  preceding  experiments  show  clearly  the  relation  which 
exists  within  certain  limits,  between  the  feeding  reaction  of  this 
organism  and  a simultaneous  reaction  to  certain  other  types  of 
stimulation.  During  stimulation  with  HCl  and  NaOH,  and  espe- 
cially with  high  temperatures  and  the  electric  current,  the  notable 
fact  is  that  the  reaction  to  food  is  strongly  persistent  under  wide 
ranges  of  intensity  of  a second  applied  stimulus;  this  is  true  to 
such  an  extent  that  under  some  conditions  the  feeding  continues 
up  to  the  point  where  the  intensity  is  so  high  that  the  stimulus 
is  destructive  to  the  organism.  These  facts  must  not  be  thought 
to  be  of  general  application,  for  evidently  mechanical  stimula- 
tion is  quite  effective  in  changing  the  reaction  to  food.  What 
the  behavior  will  be  under  two  simultaneous  stimuli  obviously 
depends  upon  the  nature  of  those  stimuli. 

It  should  be  distinctly  noted  that  in  all  the  foregoing  experi- 
ments the  chemical  as  well  as  physical  nature  of  the  food  sub- 
stance has  been  kept  constant  while  the  organism  in  its  particu- 
lar physiological  state  has  been  acted  upon  by  certain  external 
agents;  these  being  of  a sufficient  variety  to  indicate  clearly 
what  role  these  different  types  of  factors  play -in  the  relation  of 
this  animal  to  food,  and  to  serve  as  a guide  to  further  inquiry. 

We  now  have  to  see  what  changes  are  produced  in  the  feeding 
reaction  by  modifying  that  factor  which  in  the  foregoing  experi- 
ments has  been  kept  constant,^  namely,  the  food.  In  the  fol- 
lowing series  of  experiments  all  ‘the  other  conditions  will  be  kept 
constant,  or  at  least  arranged  in  such  a way  that  they  may  be 

® An  exception  to  this  might  be  taken  in  the  experiments  with  HCl  and  NaOH 
for  it  is  a question  whether  or  not  these  affect  the  chemical  character  of  the  yolk 
sufficiently  under  the  conditions  of  these  experiments  to  modify  the  number  of 
grains  eaten.  The  yolk  was  not  treated  previous  to  the  feeding;  thus  the  time 
was  so  short  and  the  dilutions  so  high  that  any  change  must  have  been  very  slight. 


RELATION  OF  BURSARIA  TO  FOOD 


29 


properly  controlled  and  accounted  for.  We  shall  attempt  to 
determine  what  the  relation  of  Bursaria  is,  to  specific  physical 
and  chemical  properties  of  the  food  itself.  First  it  will  be  deter- 
mined how  the  external  part  of  the  reaction  is  modified,  that  is, 
what  is  the  behavior  of  the  cell  in  so  far  as  this  has  to  do  with 
the  selection  of  food. 

SELECTION  OF  FOOD  AND  THE  FACTORS  CONCERNED 

The  object  of  the  experiments  described  in  the  present  section 
is  to  answer  the  question:  Can  Bursaria  discriminate  quantita- 
tive or  qualitative  differences  between  the  yolk  grains? 

When  fresh  hard  boiled  yolk  grains,  prepared  as  described 
on  page  8,  are  treated  with  different  kinds  of  water-soluble 
dyes,  the  amount  of  dye  which  is  adsorbed  by  a grain  of  yolk 
varies  with  the  kind  of  dye  used.  At  first  a considerable  number 
of  different  dyes  in  aqueous  solution  were  tested  in  a compara- 
tively rough  way;  first,  for  the  relative  amount  of  each  dye 
which  would  be  taken  up  by  the  grains  of  yolk;  second,  for  the 
rate  'at  which  the  dyes  were  adsorbed  and  the  ease  with  which 
they  could  be  washed  out  (reversibility  of  the  adsorption);  and 
third,  for  the  relative  toxicity  of  aqueous  solutions  of  these  dyes 
to  the  organisms.  Among  the  dyes  so  tested  were  fuchsin,  lyons 
blue,  methylin  blue,  eosin,  cyanin,  gentian  violet,  saffranin,  janus 
green,  Congo  red,  and  an  aqueous  solution  of  hematoxylin. 

The  results  of  the  following  experiments  on  food  selection,  in 
so  far  as  they  are  related  to  the  dye,  depend  upon  the  three 
factors  named:  (1)  The  amount  of  dye  adsorbed  (2)  The  rate 
of  the  reversible  adsorption  reaction,  and  (3)  The  relative  tox- 
icity to  Bursaria,  of  the  dye  in  aqueous  solution. 

Tt  was  quickly  found  that  certain  dyes  were  better  suited  than 
others,  for  the  particular  end  in  view.  Aqueous  solutions  of 
saffranin  and  janus  green  were  found  best  to  fulfil  the  necessary 
conditions.  Both  show  a reversible  adsorption  with  yolk,  while 
the  velocity  of  the  reversible  adsorption  is  sufficiently  low  to 
prevent  a too  rapid  washing  out  of  the  stain.  By  this  means 
one  is  able  to  control  the  amount  of  adsorbed  dye  much  more 


30 


E.  J.  LUND 


easily  than  if  it  could  be  washed  out  quickly,  and  one  is  also 
able  to  control  the  concentration  gradient  between  pure  water 
and  the  dye  adsorbed  by  the  yolk  grain.  The  toxicity  of  the 
different  dyes  varies  greatly,  and  it  was  found  that  saffranin 
and  janus  green  were  best  from  this  point  of  view  also,  since 
both  of  these  are  very  toxic  to  Bursaria  in  higher  concentrations 
but  only  slightly  so  in  lower  concentrations. 

1.  Experiments  loith  stained  and  unstained  yolk 
a.  Saffranin. 

Experiment  XII  (a).  Object,  to  test  (a)  whether  or  not  Bursaria 
will  eat  yolk  grains  which  have  adsorbed  an  appreciable  amount  of  the 
soluble  toxic  substance  saffranin  and  (b)  whether  or  not  the  amount 
of  yolk  eaten  depends,  in  this  experiment,  upon  the  amount  of  saffranin 
adsorbed. 

Equal  volumes  of  a strong  suspension  of  fresh  yolk  were  placed  in 
each  of  seven  stender  dishes  of  8 cc.  capacity.  A bright  rose-colored 
solution  of  saffranin  was  made  up  with  tap  water.  To  the  dishes 
designated  A,  B,  C,  D,  E and  F was  added  5,  4,  3,  2,  1,  and  0.5  cc.  re- 
spectively, of  this  solution,  and  mixed  thoroughly.  The  seventh  dish 
without  stain,  was  kept  as  a control.  The  suspensions  were  left  to 
settle  five  minutes,  then  decanted  and  5 cc.  tap  water  added  to  all  the 
dishes;  this  was  repeated  three  times.  The  organisms  used  were  starved 
twenty-four  hours  and  were  in  excellent  condition.  The  time  of  feed- 
ing was  fifteen  minutes  (table  13) 


TABLE  13 

Experiments  XII  {a) 


When  stronger  solutions  of  saffranin  than  that  in  A were  used, 
no  grains  were  eaten.  All  the  animals  at  the  end  of  the  experi- 
ment were  normal  and  had  not  been  injured.  The  yolk  of  dish 


i 


V 


RELATION  OF  BURSARIA  TO  FOOD 


31 


A was  now  left  to  soak  in  its  water  for  fifteen  minutes  longer, 
this  water  was  then  drawn  off  and  the  yolk  again  washed  twice 
with  water.  Thirty  individuals  from  the  same  material  as  used 
above  were  now  put  into  the  dish.  At  the  end  of  fifteen  minutes 
the  following  was  the  count: 


Experiment  XII  (b) 

3,  1,  5,  1,  3,  4,  4,  3,  9,  3,  7,  3,  0,  4,  5,  3,  6,  0,  0,  4,  1,  4,  5,  0,  0,  2,  3,  1,  3, 


Total  88  grs- 


This  indicates  that  we  may  obtain  the  same  result  whether  we 
proceed  with  a strongly  stained  yolk  and  test  successively  after 
each  washing,  or,  as  in  the  former  experiment,  by  staining  dif- 
ferent portions  of  yolk  to  different  degrees  to  begin  with.  This 
was  actually  done  in  other  experiments  not  given,  and  results 
exactly  similar  to  those  in  Experiment  XII  (a)  were  obtained. 

To  show  that  even  a considerably  stronger  medium  does  not 
injure  the  animals  seriously,  a yolk  suspension  stained  with 
saffranin  more  strongly  than  that  used  in  A of  Experiment  XII 
(a) , was  made  by  leaving  the  yolk  several  hours  in  a very  stVong 
solution  of  the  stain.  This  was  washed  out  several  times  and 
then  thirty  individuals  from  the  same  culture  material  used  in 
the  former  experiments  were  put  into  it  and  left  for  fifteen  min- 
utes. They  were  then  picked  out  and  washed  once  in  tap  water, 
and  then  transferred  to  an  unstained  yolk  suspension  for  fifteen 
minutes.  The  count  gave  the  following  (table  14): 


TABLE  14 

Experiment  XIII 


MIXTURE 

NUiMBER 

OF 

GRAINS 

EATEN  BY 

EACH  INDIVIDUAL 

Stained 

. . 0 0 

0 

0 

0 

0 

0;  0 

0 0 

0 

0 0 

0 

0 

' 0 

0 

0 

0 

0 

0^  Oj  2 

0 

o 

o 

o 

o 

o 

Unstained 

. . 0 2' 
1 

0 

o 

0 

0 

o'  0| 

0 1 

0 

3|l 

0 

0 

2 

1 

i ^ 

2 

2 

3 2 8 

0 

Oi  0^  0 0 2j  0 

1 1 ■ ! 

2 

35 


This  shows  that  they  were  not  injured  sufficiently  in  even  this 
strongly  stained  suspension  totally  to  prevent  them  from  eating 
unstained  yolk  immediately  afterward.  The  cause  of  most  of 
the  O’s  in  the  count  is  that,  as  the  toxicity  of  the  solution  in- 
creases, the  organisms  have  a tendency  to  close  up  the  oral 
apparatus  and  do  not  open  it  again  sufficiently,  within  the  next 
twenty  minutes  or  so,  to  be  able  to  take  in  the  yolk  grains.  Of 


32 


E.  J.  LUND 


course  if  yolk  which  has  been  very  strongly  stained  and  not 
washed  out  sufficiently,  is  fed,  then  the  concentration  of  the 
medium  rises  so  quickly  that  they  are  greatly  injured  or  killed. 

Now  against  the  conclusions  which  will  be  drawn  from  the  re- 
sults of  Experiment  XII  (a)  and  (b),  as  it  stands,  may  still  be 
urged  the  objection  that  the  reason  that  so  few  or  no  grains  are 
eaten,  is  because  of  what  one  might  call  a general  injury  or  stimu- 
lation of  the  cell  by  the  saffranin  which  is  rapidly  being  liberated 
into  the  water,  and  that  it  may  not  have  anything  to  do  with  a 
specific  reaction  to  the  chemical  character  of  the  food  particle  as 
such,  that  is,  to  anything  like  a sense  of  taste.”  That  this 
objection  does  not  apply  to  conditions  like  those  in  the  above 
experiment  (XII,  a and  b)  where  the  amount  of  stain  adsorbed 
even  in  dishes  A and  B is  very  little  compared  to  that  in  Experi- 
ment XIII,  may  be  shown  by  taking  the  solution  of  dish  A, 
Experiment  XII  (a),  and  placing  unstained  yolk  and  Bursariae 
in  it.  The  result  of  such  an  experiment  is  that  the  organisms 
fill  up  with  fresh  yolk,  showing  that  the  medium  in  weaker  con- 
centrations does  not  affect  the  eating  process  to  any  appreciable 
extent.  Further  proof  of  this  will  be  given  in  the*  experiments 
immediately  following,  and  also  in  experiments  to  be  given  later. 


Experiment  XIV.  To  test  whether  or  not  Bursaria  can  select  and 
eat  non-toxic  yolk  grains  from  among  toxic  ones,  when  the  two  are 
mixed.  Two  suspensions  were  made,  one  of  yolk  stained  in  saffranin 
twenty-four  hours,  then  washed  out  repeatedly,  the  other  containing 
the  same  kind  of  yolk  washed  in  the  same  way' but  not  stained.  The 
two  yolk  suspensions  were  mixed  immediately  before  the  animals  were 
placed  in  the  mixture.  Twenty  individuals  were  used.  The  time  of 
feeding  was  fifteen  minutes;  a control  of  washed  unstained  yolk  alone, 
was  kept  at  the  same  time  (table  15). 


TABLE  15 


Experiment  XIV 


MIXTURE  NUMBER  OF  GRAINS  EATEN  BY  EACH  INDIVIDUAL 


Stained 

...  0 

0 

0, 

0 0 

0 

0 

0 

1 0 

0 

0 

0 

0 

0 

X 

X 

X 

X 

X 

X 

Unstained 

Control : 

...  0 

5 

2| 

1 

si  2 

i 

1 

2 

4 

% 

0 

1 

2 

3 

2 

X 

X 

X 

X 

X 

X 

Unstained  yolk . . , 

...12 

16 

L5  4 

9 

15 

8 

9 

9 

16 

25 

7 

15 

12 

9 

9 

5 

TOTAL 

0 

29 

213 


RELATION  OF  BURSARIA  TO  FOOD 


33 


In  the  mixture  the  concentration  of  the  saffranin  rose  so  rapidly 
that  some  of  the  individuals  were  killed. This  is  indicated 
above  by  X.  Yet  even  in  such  a strong  solution,  selection  took 
place,  though  the  number  of  grains  eaten  was  small  compared 
to  the  number  in  the  control.  ^ 

Experiment  XV.  Another  sample  of  yolk  less  deeply  stained  than 
that  in  Experiment  XIV,  was  washed  out  many  times  and  mixed  with 
an  equal  quantity  of  unstained  yolk  from  the  same  sample.  Thirty 
individuals  of  the  same  material  as  used  in  Experiment  XIV,  were 
fed  for  five  minutes,  instead  of  fifteen  minutes  as  before  (table  16). 


TABLE  16 
Experiment  XV 


MIXTURE 

NUMBER  OF 

GRAINS  EATEN  BY 

EACH  INDIVIDUAL 

TOTAL 

Stained 

li  1 0 

1 

1 0 3 3 

2 

2 

O'  1 

0 

3 1 

2 

4 

4 

1 

2 

2!  t 

2 

1 

0 

0 

1 

2 

43 

Unstained 

2j  6 

7 216 

14| 

41  1 11 11 

12 

9 

7:13 

9; 

o|  6 

5 

12 

9 

5 

7 

2 10 

7 

5 

7 

3 

8 

6 

216 

A repetition  of  the  above  experiment  with  yolk  stained  a little 
more  deeply  gave  the  following  result;  twenty  individuals  used; 
fed  five  minutes  (table  17). 


TABLE  17 


NUMBER  OF  GRAINS  EATEN  BY  EACH  INDIVIDUAL 


Stained 

0 

0 

3I  1 

2 

2 

1 

1 

0 

1 

1 

1 

1 

2 

1 

3 

1 

0 

1 

3 

2 

1 

0 

Unstained i 

4 

0 

0:  1 

1 1 

4 

5 

5 

0 

4 

2 

3 

5 

3 

0 

1 

3 

0 

12 

1 

3 

25 

56 


The  control  of  Experiment  XIV  will  likewise  serve  for  Experi- 
ment XV. 

The  results  of  this  experiment  are  to  be  explained  by  the  fact 
that  the  concentration  gradient  of  the  adsorbed  toxic  saffranin 
is  relatively  low  with  i*espect  to  the  gradient  of  the  water-soluble 
yolk  substance  to  which  Bursaria  reacts  in  a strongly  positive 
manner.  Of  course  one  is  not  to  suppose  that  it  is  the  relative 
molecular  concentration  gradient  alone  that  determines  the  re- 

The  cytolytic  action  of  saffranin  is  in  some  ways  more  marked  than  that  of 
janus  green.  The  character  of  its  reversible  adsorption  reaction  also  makes  it 
less  suited  for  use  in  experiments  of  this  kind  than  janus  green,  as  will  appear 
from  results  with  the  latter. 


THE  JOURNAL  OF  EXPERIMENTAL  ZOOLOGY,  VOL.  16,  NO.  1 


34 


E.  J.  LUND 


suit.  What  is  meant  in  this  case  by  concentration  gradient,  is 
the  molecular  concentration  plus  the  specificity  of  the  substance, 
that  is,  in  anthropomorphic  terms  we  should  say  “the  kind  of 
taste’’  which  the  substance  has.  That  the  specific  nature  of 
the  substance  is  one  factor  in  determining  the  result,  is  shown 
by  a comparison  of  the  results  of  numerous  experiments  with 
saffranin,  janus  green,  hematoxylin,  and  especially  other  less 
toxic  stains,  like  Congo  red  (cf.  what  follows). 

h.  Janus  green.  A considerable  number  of  experiments  have 
been  carried  out  using  this  substance,  with  the  same  general 
results  as  those  obtained  with  saffranin.  It  is  better  adapted 
to  bring  out  the  phenomenon  of  selection  than  saffranin,  causing 
a sharp  discrimination  by  Bursaria;  small  quantities  adsorbed 
by  the  grains  are  sufficient  to  bring  about  rejection.  The  fol- 
lowing experiments  show  some  of  the  relations.  a 


Experiment  XVI  (a)  and  (b) . Yolk  was  stained  in  janus  green  twenty- 
four  hours  then  soaked  in  tap  water  and  washed  repeatedly.  A portion 
of  the  same  kind  of  yolk  soaked  and  washed  in  the  same  way  but  not 
stained,  was  used  as  a control  and  for  mixing  with  the  stained  yolk. 
A few  minutes  before  the  experiment  equal  quantities  of  the  stained 
and  unstained  yolk  suspension  were  mixed  in  dish  A.  A second  quan- 
tity of  the  unstained  yolk  suspension  of  a concentration  equal  to  the 
sum  of  those  in  dish  A was  placed  in  dish  B.  Twenty  individuals  were 
placed  in  each  dish  and  left  to  feed  twenty  minutes  (table  18  a). 


TABLE  18  (a) 
Experiment  XVI  (a) 


MIXTURE,  DISH  A 


NUMBER  OF  GRAINS  EATEN  BY  EACH  INDIVIDUAL 


Stained 

0 0 

0 

0 

0 

0 

1 

0 

0 

0 

0 

0 

0 

0 

0 

0 

X 

X 

X 

X 

X 

Unstained 

5 3 

1 

6 

2 

4 

1 

4 

5 

1 

5 

1 

4 

4 

1 

X 

X 

X 

X 

X 

Control, 

Dish  B : 

Unstained 

912i21 

8 

20112 

15 

19 

13 

17 

1413 

15 

12 

19 

17 

14 

7 

8 

3 

TOTAL 

0 

47 


268 


The  yolk  in  both  dishes  was  now  washed  twice  and  the  experi- 
ment repeated  with  control.  Time  of  feeding  fifteen  minutes. 
The  count  is  shown  in  table  18  b. 

In  (a)  the  solution  had  become  sufficiently  strong  to  affect 
five  of  the  animals  (X),  so  that  they  could  not  be  recovered  for 


RELATION  OF  BURSARIA  TO  FOOD 


35 


TABLE  18  (b) 
Experiment  XVI  (b) 


MIXTURE,  DISH  A 


Stained 

Unstained. . 
Control, 
Dish  B: 
Unstained. . 


NUMBER  OF  GRAINS  EATEN  BY  EACH  INDIVIDUAL 


0 

i 

0 

0 

1 

0 

I 

0 

0 

0 

0 

0 0 

0 

0 

0 

0 

0 

1 

0 

0 

0 

1 

0 

0 

0 

0 

0 

0 

0 

1 

1 

2 0 

1 

2 

2 

4 

0 

0 

1 

0 

1 

3 

19 

16 

8 

17 

7 

13 

10 

11 

12 

512 

1 

4 

5 

12 

15 

15 

16 

7 

10 

13 

8 

216 

the  count.  The  smaller  number  of  grains  eaten  in  (b)  by  those 
in  dish  A is  due  in  part  to  the  shorter  time  of  feeding  but  more 
to  the  fact  that  the  unstained  yolk  grains  had  by  this  time  ad- 
sorbed some  of  the  liberated  janus  green  from  the  stained  yolk 
grains  (see  Experiment  XVIII,  p.  36). 


Experiment  XVII  (a)  and  (b).  The  results  of  this  experiment  are 
given  to  show  that  Bursariae  from  two  different  cultures  may  show 
different  reactions  in  selection  experiments.  In  part  (a)  material  was 
used  from  one  wild  culture,  while  in  part  (b)  material  from  a differ- 
ent one  was  used.  Both  were  starved  twenty-four  hours  before  using 
them.  All  other  conditions  were  alike.  Time  of  feeding  fifteen  minutes 
(table  19). 


TABLE  19 


MIXTURE,  DISH  A 


Experiment  XVII 


NUMBER  OF  GRAINS  EATEN  BY  EACH  INDIVIDUAL 


Stained 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

Unstained 

0 

4 

0 

0 

0 

0 

0 

0 

0 

0 

1 

1 

1 

1 

1 

0 

0 

0 

0 

0 

9 

Control:  dish  B, 

(a) 

Unstained 

5 

4 

6 

2 

4 

7 

6 

3 

9 

3 

8 

3 

3 

4 

3 

2 

7 

6 

8 

99 

Stained 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

Unstained 

3 

4 

0 

2 

10 

3 

3 

2 

7 

6 

1 

6 

6 

0 

8 

2 

1 

0 

5 

1 

70 

Control:  dish  B, 

(b) 

Unstained 

2 

4 

13 

1 1 

9 

5 

7 

9 

2 

4 

1 

10 

6 

15 

13 

6 

11 

9 

1 

6 

7 

1 

148 

In  such  experiments  as  these  it  was  found  that  occasionally  an 
individual  had  eaten  a stained  grain  along  with  the  unstained 
ones,  but  this  happened  very  seldom  in  any  of  the  experiments 
with  janus  green. 


36 


E.  J.  LUND 


Experiment  XVIII.  The  two  dishes  A and  B in  Experiment  XVII 
(b)  were  left  standing  for  two  hours;  then  they  were  washed  once  and 
tested  with  the  same  material  used  in  Experiment  XVII  (b),  in  order 
to  show  the  effect  of  the  adsorption  of  the  liberated  stain  by  the  un- 
stained grains  mixed  with  the  stained  ones.  Time  of  feeding  fifteen 
minutes  (table  20). 


TABLE  20 

Experiment  XVIII 


MIXTURE,  DISH  A 


NUMBER  OF  GRAINS  EATEN  BY  EACH  INDIVIDUAL 


Stained 

0 

0 

0 

0 

0 

0 

o' 

! 0 

0 

0 

0 

0 

0 0 

0 

0 

0 

0 

0 

0 

Unstained ' 

0 

2 

0 

7 

2 

0 

1 

4 

0 

1 

0 

0 

0 2 

0 

2 

0 

0 

0 

0 

21 

Control:  dish  B, 

i 

Unstained 

18 

23 

8 

15 

13 

7 

0 

9 

1215 

4 

17 

1612 

15 

15| 

11 

o| 

8 

10 

228 

This  shows  that  when  the  janus  green  yolk  is  left  with  the  un- 
stained yolk  for  some  time,  the  liberated  stain  from  the  janus 
green  yolk  is  adsorbed  by  the  unstained  yolk  grains,  and  as  a 
result  the  latter  are  not  eaten  so  readily.  If  the  mixture  is  left 
standing  too  long  and  then  rinsed  in  tap  water,  then  no  grains 
are  eaten.  Bursaria  can  react  to  such  small  quantities  of  ad- 
sorbed janus  green  that  the  amount  adsorbed  cannot  be  dis- 
tinguished by  the  eye,  when  the  unstained  yolk  grains  mixed 
with  the  stained  ones  are  examined. 


Experiment  XIX.  To  prove  that  the  solution  of  the  janus  green 
which  is  produced  by  the  liberation  of  the  stain  from  the  stained  yolk, 
does  not  even  in  quite  strong  concentrations  prevent  the  eating  of 
fresh  yolk  placed  in  it,  the  result  of  one  test  out  of  a considerable  num- 
ber made  at  different  times,  is  given.  A solution  was  drawn  off  from 
janus  green  stained  yolk  used  in  an  experiment  in  which  no  grains  of  the 
mixture  had  been  eaten,  after  standing  for  some  time.  To  this  solution 
was  added  unstained  yolk.  Ten  individuals  were  tested  (table  21). 


TABLE  21 


Experiment  XIX 


NUMBER  OP  GRAINS  EATEN  BY  EACH 
INDIVIDUAL 


Fresh  yolk  in  janus  green  solu- 
tion, dish  A 

8 

3 

6 

3 

1 

0 

! 

3 

0 

1 

1 

3 

Control,  fresh  yolk  in  tap  water, 
dish  B 

5 

15 

1 

5 

0 

6 

5 ; 

3 

1 

TOTAL  . 


0 


0 


44 


RELATION  OF  BURSARIA  TO  FOOD 


37 


It  is  evident  that  the  solution  of  janus  green  drawn  from  the 
mixed  suspension,  and  produced  by  the  liberation  of  the  stain 
from  the  stained  yolk  grains  of  the  mixture  which  was  not  eaten, 
did  not  now  prevent  the  animals  from  eating  unstained  yolk 
grains  which  were  placed  in  it;  hence  it  was  not  the  stain  in 
solution  which  prevented  the  eating  of  the  stained  grains  of  the 
mixture  from  which  the  solution  was  drawn;  but  the  stain  which 
was  adsorbed  by  the  yolk  grains  of  the  mixture  and  diffused 
from  them.  Many  such  similar  tests  were  carried  out  giving 
the  same  result.  This  does  not  mean  that  the  solution  apart 
from  the  yolk  grain  with  its  adsorbed  dye,  may  not  affect  the 
result  of  the  feeding,  for  in  higher  concentrations  the  solution 
apart  from  the  stain  upon  the  grain  does  affect  the  feeding  proc- 
ess. In  solutions  of  lower  concentrations  of  the  appropriate  dye 
the  chemical  nature  of  the  grain  along  with  the  amount  of  dye 
adsorbed,  are  the  essential  factors  determining  the  number  of 
grains  which  will  be  eaten. 

c.  Hematoxylin.  To  show  further  that  the  specificity  of  the 
toxic  agent  plays  a large  part  in  determining  whether  or  not  yolk 
will  be  eaten,  the  following  experiments  are  given.  It  will  be 
noted  that  in  this  case  we  have  a substance  which  has  a very 
different  effect  upon  the  cell  and  its  relation  to  food,  from  that 
produced  by  the  substances  thus  far  dealt  with.  The  solution 
in  this  case  may  be  made  very  deep  brown  while  the  grains  are 
also  stained  deeply,  and  yet  the  yolk  grains  are  eaten  even  in 
solutions  which  kill  the  animals  if  they  remain  in  it  more  than 
three  or  four  minutes. 

Experiment  XX:  Table  22  (a)  and  (6).  The  same  quantity  of  yolk 
was  added  to  each  of  nine  dishes  of  8 cc.  capacity,  each  containing 
equal  amounts  of  tap  water.  The  dishes  were  numbered  1,  2,  3,  and 
so  forth.  To  these  were  added  diverse  quantities  of  the  0.5  per  cent 
aqueous  solution  of  hematoxylin  by  drops,  as  given  in  the  tables;  time 
of  feeding  ten  minutes. 

This  experiment  was  repeated  with  the  same  suspensions  at 
the  end  of  one  hour;  time  of  feeding  fifteen  minutes  (table  22  6). 
The  individuals  which  died  before  the  count  was  made  are  de- 
noted by  X.  The  tables  show  that  although  the  solutions,  espe- 


38 


E.  J.  LUND 


cially  ill  higher  concentration,  are  very  injurious,  the  organisms, 
nevertheless,  eat  the  grains  of  yolk.  After  one  or  more  hours 
the  grains  become  stained  deeply.  This  was  the  case  in  table 
22  (b).  The  increase  in  the  length  of  time  of  feeding  (i.e.,  the 
time  the  animals  were  left  in  the  solution)  is  the  cause  of  the 
high  mortality  in  table  22  (b). 


NUMBER  OF  DISH 


Number  of  drops  of  ^ 
per  cent  aq.  hema- 
toxylin   


Number  of  grains 
eaten  by  each  in- 
dividual   


Total. 


TABLE  22  (a) 
Experiment  XX 


■ ^ 

, 1 

2 

3 

4 

5 

6 

7 

8 

9 

1 

4 

8 

12 

16 

20 

24 

28 

50 

0 

0 

0 

0 

2 

1 

4 

7 

7 

4 

1 

2 

3 

4 

6 

0 

G 

6 

2 

0 

11 

7 

3 

X 

10 

2 

5 

4 

1 

3 

7 

X 

11 

2 

4 

0 

1 

lost 

1 

1 

X 

13 

0 

0 

0 

1 

11 

1 

X 

8 

! 4 

1 

1 

2 

7 

4 

X 

14 

G 

7 

3 

4 

0 

4 

X 

G 

0 

3 

0 

13 

2 

2 

X 

6 

4 

0 

7 

X 

4 

X 

X 

8 

28 

23 

17 

38+ 

40 

32+ 

(7+) 

89 

TABLE  22  (b) 


NUMBER  OF  DISH 

1 

2 

3 

4 

5 

6 

7 

8 

9 

Number  of  drops  of 

§ per  cent  aq.  hema- 

toxylin  

4 

8 

12 

16 

20 

24 

28 

50 

0 

I 

3 

1 

0 

1 

1 

0 

X 

X 

11 

5 

1 

1 

2 

X 

3 

X 

X 

8 

0 

2 

0 

1 

X 

0 

X 

X 

7 

3 

3 

4 

0 

X 

2 

X 

X 

11 

Number  of  grains  ^ 

2 

0 

3 

0 

X 

X 

X 

X 

14 

eaten  by  each  in- 

5 

6 

0 

0 

X 

X 

X 

X 

8 

dividual  

4 

0 

0 

X 

X 

X 

X 

X 

6 

5 

3 

0 

X 

X 

X 

X 

X 

8 

3 

1 

0 

X 

X 

X 

X 

X 

13 

3 

X 

X 

X 

X 

X 

X 

X 

7 

Total 

33 

17+ 

8+ 

4+ 

1+ 

5+ 

- 

- 

93 

. RELATION  OF  BURSARIA  TO  FOOD 


39 


That  the  yolk  grains  are  eaten,  though  to  a less  extent,  even 
after  having  been  left  in  the  solution  of  50  drop  concentration 
for  three  and  one-half  hours,  is  shown  by  the  following:  Feeding 
was  limited  to  three  minutes,  which  in  part  accounts  for  the 
comparatively  small  number  of  grains  eaten.  Ten  individuals 
were  used.  The  count  gave  4,  2,  1,  6,  1,  1,  2,  0,  0,  1,  a total  of 
18  grains  of  the  deeply  stained  yolk.  When  this  deeply  stained 
yolk  in  the  50  drop  concentration  of  hematoxylin  was  washed 
four  times  in  tap  water  and  tested  again;  the  following  count 
was  made:  11,  13,  14,  6,  5,  2,  12,  2,  4,  5,  a total  of  74 
grains. 

These  tests  show  (a)  that  although  a dye  may  be  toxic  to 
Bursaria,  it  may  nevertheless  not  affect,  to  any  great  extent,  the 
functioning  of  the  feeding  mechanism  in  the  talj;ing  in  and  swal- 
lowing of  the  food,  though  (b)  with  some  dyes  total  rejection 
of  the  food  may  take  place,  when  the  concentration  is  so  low 
that  it  has  only  a comparatively  slight  cytolytic  effect.  The 
former  condition  is  shown  to  a less  marked  extent  in  the  experi- 
ments with  saffranin  than  in  the  experiments  with  hematoxylin  ; 
while  the  latter  condition  is  illustrated  by  the  results  with  janus 
green.  This  seems  then  also  to  strongly  suggest  that  different 
substances  may  affect  different  parts  of  the  cell  differently. 
Corroborative  evidence  upon  this  point,  which  it  would  be  out 
of  place  to  consider  here,  has  been  obtained  from  observations 
showing  that  the  localization  of  the  beginnings  of  cytolysis  of 
the  cell  body  of  Bursaria  may  differ  with  the  particular  nature 
of  the  toxic  agent  employed. 

d.  Congo  red.  Another  stain  which  is  adsorbed  readily  is  Congo 
red.  This,  however,  unlike  hematoxylin,  can  only  be  washed 
out  in  part,  that  is,  its  adsorption  reaction  is  not  completely 
reversible.  Also  since  this  dye  is  not  as  toxic  as  saffranin  or 
janus  green,  a large  quantity  of  the  stain  may  be  adsorbed  and 
yet  not  appreciably  affect  the  number  of  grains  eaten,  as  is 
shown  by  the  following  experiment. 

Experiment  XXL  The  yolk  was  stained  twenty  minutes  in  a strong 
aqueous  solution  of  the  dye.  Time  of  feeding  twenty  minutes.  Thirty 
were  used  (table  23). 


40 


E.  J.  LUND 


TABLE  23 
Experiment  XXI 


NUMBER  OF  GRAINS  EATEN  BY  EACH  INDIVIDUAL 

TOTAL 

Congo  red,  dish  A i j 

Stained 7 sj  6 

Control  dish  B 1 1 

L^nstained if  sj  7 

1 

3 

7 

6 

8 

2 

5 

6 

1 

6 

2! 

5 

5 

8 

4 

7 

4 

5 

3 

1 

I 

3 

2 

i 

5|  6 

4 

7 

4 

1 

0 

5 

5 

8 

1 

& 

! 

1^3 
f 710 

2 

5 

4 

6 

6 

5 

5 

9 

5 

4 

4 

1 

1 7 

115 

179 

In  this  case  we  have  a comparatively  low  concentration  gradient 
of  the  dye,  together  with  a low  toxicity  and  hence  the  compara- 
tively small  difference  in  the  readiness  with  which  Bursaria  eats 
the  stained  and  unstained  yolk. 

e.  Sudan  III.  To  show  that  an  adsorbed  substance  which  is 
insoluble  in  the  medium  has  no  determining  effect  upon  the 
feeding  and  food  selection,  Sudan  III  was  employed.  This  sub- 
stance is  insoluble  in  water  but  soluble  in  ethyl  alcohol  and  fats. 

Experiment  XXII.  Fresh  yolk  was  stained  in  an  80  per  cent  alco- 
holic solution  of  Sudan  III  for  a short  time.  It  was  then  dried  in  an 
oven  at  21° C.  for  twenty-four  hours.  A control  of  fresh  yolk  was  also 
kept.  The  organisms  were  fed  twenty  minutes.  The  yolk  takes  on 
a very  deep  color  with  this  stain. 


Sudan  III,  stained 

yolk 

Control,  unstained. 


TABLE  24 

Experiment  XXII 


NUMBER  OF  GRAINS  EATEN  BY  EACH  INDIVIDUAL 


13 


18 

19 

0 

15 

3 

5 

7 

18 

25 

15 

5 

7 

8 

9 

17 

18 

i 

12 

1 

3 

26 

3 

4 

14 

7 

15 

17 

22 

6 

28 

20 

6 

9 

3 

29 

18 

13 

10 

8 

24 

243 

274 


It  is  evident  that  the  insoluble  Sudan  III  had  no  appreciable 
effect  upon  the  food  reaction.  Mixtures  of  these  showed  no 
difference  in  the  amounts  of  the  two  kinds  of  yolk  eaten. 

/.  Stale  yolk. 

Experiment  XXIII.  Mixtures  of  fresh  and  stale  yolk  could  not  be 
used  since  the  grains  of  the  two  kinds  of  yolk  were  visibly  indistinguish- 
able. One  experiment  is  given.  The  stale  yolk  was  four  weeks  old 
while  the  control  was  freshly  prepared;  both  were  of  the  same  con- 
centration (table  25). 


RELATION  OF  BURSARIA  TO  FOOD 


41 


TABLE  25 

Experiment  XXIII 


Stale  yolkl 

Control,  fresh  yolk. 


NUMBER  OF  GRAINS  EATEN  BY  EACH  INDIVIDUAL 


o’  2 

5 

1 

0 

3 

8 

2 1 

1 

4 

0 

6 

1 

5 

1 

3 

1 

1 

8 4 

7 

2 

11 

11 

4 

4I  9 

I 

3 

0 

7 

9 

5 

2 

2 

6 

1 

0 

48 

96 


A difference  is  plainly  evident.  Other  experiments  show  greater 
or  less  difference,  depending  upon  the  conditions. 

2.  The  basis  for  and  nature  of  food  selection  in  Bursaria,  as  shown 
by  the  foregoing  and  other  experiments 

It  must  be  remembered  that  in  any  such  experiments  as  the 
foregoing  the  relation  to  food  is  in  some  ways  an  entirely  new 
one  to  the  organism.  Yet  it  must  be  insisted  upon  that  the 
yolk  used  in  these  experiments  is  assimilable  by  the  organisms 
(a  fact  which  will  be  considered  at  length  in  a later  paper)  and 
especially  that  whatever  the  mechanism  of  feeding  and  selection 
in  nature  is,  it  must  be  the  same  one  which  is  brought  into  action 
in  these  experiments.  Hence  the  criticism  imagined  above  would 
appear  to  have  no  importance  for  the  question  under  consider- 
ation here.  In  fact,  it  is  to  be  believed  that  so  far  as  these 
experiments  are  concerned,  they  are  only  a more  strongly  empha- 
sized condition  of  what  we  find  in  nature  and  that  they  picture 
to  us,  so  far  as  they  go,  the  actual  condition  of  the  food  relation 
of  Bursaria  in  its  native  culture. 

We  may  state  the  results  briefly  in  the  following  way:  First: 
Yolk  grains  are  rejected  if  the  soluble  adsorbed  toxic  substance 
makes  with  the  medium  a sufficiently  steep  concentration  gra- 
dient. If  this  gradient  is  low  relative  to  that  of  the  yolk-soluble 
substance,  to  which  Bursaria  reacts  positively,  then  the  organism 
may  eat  the  stained  yolk,  other  conditions  being  equal.  Second : 
(a)  Whether  Bursaria  will  eat  stained  yolk  grains  or  reject  them 
depends  also,  along  with  the  steepness  of  the  concentration  gra- 
dient, upon  the  specific  chemical  properties  of  the  adsorbed  sub- 


42 


E.  J.  LUND 


stance  in  question,  and  furthermore  (b)  the  substance  by  virtue 
of  its  chemical  properties  has,  at  least  in  some  cases,  a specific 
action  upon  the  mechanism  of  feeding  and  selection,  as  is  shown 
by  a comparison  of  the  results  of  the  experiments  with  hema- 
toxylin, saffranin  and  janus  green.  Additional  evidence  obtained 
from  observations  upon  the  phenomena  of  cytolysis  in  Bursaria 
also  points  to  the  correctness  of  this  conclusion.  A familiar  in- 
stance of  a similar  nature  is  the  casting  off  of  the  peristome  by 
Stentor  when  stimulated  or  injured  by  chemicals.  Another  in- 
stance is  the  fact  found  by  Jennings  that  the  anterior  end  in 
Paramecium  is  more  sensitive  to  mechanical  stimulation  than  are 
other  parts  of  the  body. 

That  in  feeding  experiments  with  the  Protozoa  it  is  difficult 
to  discriminate  closely  between  the  effects  of  the  medium  and 
those  of  the  food  substance  itself  is  obvious,  since  (a)  the  amount 
eaten  depends  upon  so  many  factors  other  than  the  nature  of 
food  and  (b),  since  the  organism  selects  on  a chemical  basis, 
which  involves  a soluble  substance  or  substances  diffusing  into 
the  medium  from  the  food  particle,  hence  necessarily  involving 
the  external  medium  to  a greater  or  less  extent.  It  is  of  course 
clear  that  differences  in  certain  physical  characters  of  food  may 
likewise  determine  whether  or  not  it  will  be  eaten.  This  is  shown 
most  simply  by  objects  which  are  too  large,  such  as  large  yolk 
grains  and  large  individuals  of  Stentor,  which  cannot  be  swal- 
lowed. 

From  all  the  facts  found  from  experiments  upon  food  selection 
by  Bursaria,  there  is  no  evidence  that  active  selection  is  based 
upon  either  ^^size,  weight,  form  or  surface  texture’’  or  any  com- 
bination of  these,  except  in  so  far  as  simple  mechanical  condi- 
tions would  make  them  effective.  All  the  facts  show  clearly 
that  the  chemical  nature  of  the  food  is  the  property  upon  which 
the  power  of  discrimination  by  Bursaria  depends.  Hence  I find 
no  evidence  from  Bursaria  to  support  Schaeffer’s  contention  that 
“Stentor  selects  its  food  upon  a tactual  basis  and  apparently 
not  upon  a chemical  one”  and  that  “Stentor  reacts  in  selecting 
food,  to  physical  properties  only  or  chiefly,  and  not  to  chemical 
properties”  (Schaeffer  ’10,  page  131).  On  the  other  hand,  the 


RELATION  OF  BURSARIA  TO  FOOD 


43 


facts  which  have  been  found  in  this  connection  are  in  agreement 
with  the  results  and  conclusions  so  far  as  they  have  been  worked 
out  by  Metalnikow  (^12)  for  Paramecium. 

THE  RELATION  OF  BURSARIA  TO  DIGESTIBLE  AND  NON-DIGESTIBLE 

SUBSTANCES 

1 . The  external  relations 

Many  substances  which  are  in  the  ordinary  sense  chemically 
indifferent  to  the  organism  are  likewise  eaten,  though  generally 
in  small  quantities.  Among  these  are  cinnabar,  carbon  black, 
Chinese  ink,  powdered  aluminium  and  the  like.  The  relation  of 
Bursaria  to  this  class  of  substances  is  however  strikingly  differ- 
ent inside  of  the  cell  and  to  a large  extent  outside,  when  com- 
pared to  that  relation  in  the  case  of  digestible  and  assimilable 
ones.  The  fact  that  some  comparatively  indifferent  substances 
like  the  above,  are  eaten  does  not  affect  our  conclusion  drawn 
above,  as  to  the  paramount  importance  of  the  chemical  prop- 
erties of  the  food  in  food  selection.  Chinese  ink  contains  some 
mucilaginous  matter  which  as  my  own  observations  have  shown 
me,  is  reacted  to  positively  by  Bursaria  and  hence  the  ink  is 
quite  readily  eaten.  Carmine  is  a similar  substance  which  though 
generally  taken  to  be  insoluble  in  water,  is  in  fact  sufficiently 
soluble  clearly  to  affect  the  feeding  reactions  of  Bursaria.  Fur- 
thermore, the  fact  that  a substance  may  be  insoluble  does  not, 
of  course,  prove  that  the  stimulus  from  it  is  not  a chemical  one, 
for  it  is  probable,  that  with  such  substances  as  aluminium,  cata- 
lytic or  other  specific  chemical,  or  even  physical  reactions  depend- 
ent upon  the  chemical  properties  of  the  substance,  are  produced 
by  contact  with  the  plasma  membranes.  The  possible  variety 
of  interactions  of  the  cell  with  different  kinds  of  substances  when 
considered  in  this  order  of  magnitude  may  of  course  be  very 
large. 

As  regards  the  eating  of  non-digestible  substances,  powdered 
aluminium  may  serve,  in  one  way,  to  illustrate  the  external  rela- 
tions. If  a large  number  of  individuals  are  put  into  a suspension 
of  aluminium,  often  few  if  any  will  eat  any  of  the  particles  of 


44 


E.  J.  LUND 


aluminium  and  those  that  do  eat  it  generally  take  in  only  a 
small  quantity.  This  is  also  true  of  Sudan  III  and  of  carboD 
black.  The  quantity  eaten  varies  with  the  conditions  in  a 
similar  way,  as  previously  set  forth  for  yolk.  Now  if  fresh  yolk 
grains  are  added  to  the  suspension  of  aluminium  the  animals 
will  often  quickly  fill  up  with  yolk,  but  in  this  case  flakes  of 
aluminium  become  attached  to  the  yolk  particles  and  hence  often 
considerable  quantities  of  the  metallic  aluminium  are  passed  into 
the  body  along  with  the  yolk.  Sometimes  the  quantity  of  yolk 
eaten  in  such  a mixed  suspension  is  less  than  that  in  the  control. 
This  serves  to  illustrate  the  sort  of  equilibrium  which  exists 
between  the  organism  and  the  kinds  of  substances  in  suspension, 
partly  determining  the  amount  of  food  and  other  substances 
eaten. 

2.  The  internal  relations 

It  was  interesting  to  find  that  Bursaria  possesses  what  I shall 
call  an  internal  compensating  reaction  to  those  substances  which 
are  eaten  to  some  extent,  but  are  not  digestible,  such  as  Sudan 
III,  Chinese  ink,  powdered  aluminium,  and  so  forth.  This  com- 
pensating reaction  makes  up  to  some  extent,  in  the  economy 
of  the  organism”  for  the  lack  of  a perfect  discrimination  between 
indigestible  (Tasteless^  substances  and  those  which  can  serve  as 
food.  It  is  shown  by  the  fact  that  indigestible  substances  are 
eliminated  from  the  cell  usually  a long  time  before  the  digestion 
of  a similar  quantity  of  food  is  completed.  This  may  be  shown 
with  Sudan  III.  The  results  of  the  experiments  are,  for  the 
sake  of  brevity,  given  by  curves. 

Experiment  XXIV:  Figure  4.-  Three  sets  of  twenty-four  individuals 
each  were  fed  Sudan  III,  cold-ether-extracted  yolk,  and  fresh  yolk 
respectively.  They  were  placed  two  in  each  watchglass  containing  tap 
water  in  moist  chambers,  and  examination  in  this  case  was  made  at 
the  end  of  three,  seven,  and  twenty-tw'o  hours.  Points  on  the  abscissae 
indicate  the  length  of  time  in  hours  after  feeding,  while  points  on  the 
ordinates  show  the  number  of  individuals  which  had  extruded  Sudan 
III  (curve  T)  in  the  time  intervals  between  the  examinations,  or  in 
the  case  of  extracted  yolk  (curve  B)  and  fresh  yolk  (curve  C)  the  nu- 
ber  of  individuals  that  had  lost  all  trace's  of  food.  In  this  experiment 
the  observations  were  not  sufficiently  frequent' to  bring  out  the  actual 


'V' 


I i\ 


* I 


RELATION  OF  BURSARIA  TO  FOOD 


45 


Fig.  4 Experiment  XXIV.  Curve  A represents  the  course  of  total  extrusion 
of  Sudan  III;  curve  B,  that  of  complete  digestion  of  cold-ether  extracted  yolk; 
curve  C,  course  of  complete  disappearance  of  fresh  fat-containing  yolk. 

course  of  the  extrusion  of  Sudan  III  or  of  the  disappearance  of  the 
yolk  from  the  cytoplasm,  but  it  will  be  noted  that  the  great  difference 
appears  in  the  observation  at  the  end  of  seven  hours.  At  the  end  of 
twenty-two  hours  A had  no  traces  of  Sudan  III,  still  had  six  and 
C had  ten  individuals  with  food. 

This  early  extrusion  of  indigestible  substances  is  considered 
in  more  detail  in  connection  with  Chinese  ink  in  the  followingd^ 

Experiment  XXV.  Two  sets  of  forty-eight  individuals  each  were 
fed,  one  with  cold-ether-extracted  yolk,  the  other  with  Chinese  ink. 
Examination  of  the  cell  content  was  made  at  hour  intervals  as  indicated 
by  numbers  on  the  basal  abscissa.  Ordinates  indicate  the  number  of 
individuals  which  had  extruded  all  the  ink  content  in  the  time  indi- 
cated (curve  A).  In  curve  B the  ordinates  represent  the  number  of 
individuals  fed  extracted  yolk  in  which  yolk  had  disappeared  at  the 
end  of  the  time  indicated  by  the  abscissa.  Chinese  ink  in  suspension 
is  much  more  readily  eaten  than  carbon  or  aluminium  and  is  therefore 
more  convenient.  This  is  to  be  explained  by  the  fact  that  there  are 
present  mucilaginous  soluble  substances  in  the  Chinese  ink  which  serve 
as  agents  inducing  a more  positive  feeding  reaction  and  are  possibly 
of  some  slight  food  value.  The  ink  was  not  found  to  be  injurious  to 
the  animals.  The  greater  part  of  the  ink  is  thrown  out  quite  early 
while  slight  traces  may  remain  for  some  time  longer.  The  time  dur- 
ing which  the  ink  was  retained  was  taken  to  end  when  the  last  trace 

In  order  to’  obtain  satisfactory  results  with  such  substances  as  Sudan  III 
and  aluminium  in  aqueous  suspension  the  adsorbed  gases  should  be  driven  off 
before  feeding. 


46  E.  J.  LUND 


Fig.  5.  Experiment  XXV.  Curve  A represents  the  course  of  extrusion  of 
Chinese  ink  by  forty-eight  individuals;  curve  B that  of  complete  digestion  of  a 
similar  quantity  of  cold-ether  extracted  yolk  by  another  set  of  forty-eight  in- 
dividuals from  the  same  culture. 

had  been  eliminated;  curve  A does  not  therefore  represent  the  actual 
time  at  which  the  greater  part  of  the  ink  was  extruded  but  should  have 
its  maxima  farther  to  the  left  than  shown.  This  statement  applies  to 
all  the  extrusion  curves  which  are  given. 

In  figure  5 curve  B,  is  that  of  complete  digestion;  no  extrusion 
of  the  extracted  yolk  took  place  in  this  experiment. 

After  it  had  been  thus  shown  that  ink  fed  alone  to  one  set  of 
individuals  was  extruded  long  before  digestion  is  completed  of 
a similar  amount  of  extracted  yolk  fed  to  another  set,  experi- 
ments were  carried  out  to  test  what  the  reaction  would  be  if 
both  ink  and  extracted  yolk  were  fed  to  the  same  individuals 
at  the  same  time.  The  following  two  experiments  are  given  to 
bring  out  the  facts  in  a quantitative  way. 

Experiinent  XXVI.  Fifty-four  individuals  were  first  fed  Chinese  ink 
and  immediately  afterwards  fed  with  cold-ether-extracted  yolk.  Two 
individuals  were  placed  in  each  watch-glass  containing  5 cc.  of  tap 


RELATION  OF  BURSARIA  TO  FOOD 


47 


water  and-  kept  in  moist  chambers.  Records  were  taken  noting  the 
presence  or  total  absence  of  ink  and  presence  or  completion  of  digestion 
of  the  extracted  yolk  at  one  hour  intervals  beginning  with  three  and 
one-half  hours  up  to  twelve  hours  after  feeding;  three  more  records 
were  taken  at  twenty-four,  thirty-three  and  forty-eight  hours.  The 
results  are  expressed  in  curves  in  figure  6.  Curve  A represents  the 
extrusion  of  ink;  curve  B,  that  of  complete  digestion  of  yolk. 

It  is  seen  from  the  relation  of  the  curves  that  even  in  this 
case  the  ink  is  extruded  before  digestion  of  the  extracted  yolk 
is  complete,  provided  that  a sufficient  quantity  of  yolk  has  been 
eaten. 

It  was  noted  that  a short  time  after  the  ink  had  been 
eaten  it  became  assembled  into  one  or  several  rather  definite 
lumps.  This  takes  place  before  extrusion.  Closer  observation 
further  revealed  the  fact  that  when  ink  particles  came  to  be 
included  in  vacuoles  containing  yolk  they  were  not  extruded 
until  the  food  of  those  vacuoles  had  been  digested,  while  those 
which  were  not  included  by  the  yolk  vacuoles  were  very  soon 
extruded.  This  fact  can  readily  be  made  out  while  one  follows 
such  experiments  as  Experiment  XXVI  above.  Bursaria  there- 
fore has  a power  of  simultaneous  selective  extrusion  of  the  con- 
tents of  different  vacuoles  as  well  as  a power  of  selection  in'the 
feeding  process.  This  mechanism  obviously  compensates  for  the 
lack  of  a perfect  discriminative  and  selective  function  of  the 
oral  apparatus. 

The  results  of  an  experiment  (fig.  7)  where  these  facts  were 
taken  into  account  for  the  purpose  of  expressing  them  in  a graphic 
way  in  curves,  is  given  in  the  following  experiment.  A control 
for  comparison  was  also  kept  in  this  case  (fig.  7,  curve  C). 

Experiment  XXVII.  Forty-eight  individuals  were  used  in  each  of 
both  the  experiment  and  control.  The  control  (curve  C,  fig.  7)  which 
was  fed  ink  only,  shows  a sharp  early  maximum  of  extrusion  from 
five  and  one-half  to  seven  and  one-half  hours  after  feeding,  with  three 
or  four  individuals  retaining  traces  of  ink  as  long  as  ten  and  one-half 
to  twelve  hours.  Curve  A represents  the  extrusion  of  ink  in  the  forty- 
eight  individuals  fed  both  ink  and  extracted  yolk.  It  shows  two  max- 
ima exactly  similar  to.  those  of  curve  A in  Experiment  XXVI.  Curve 
B (fig.  7)  represents  the  course  of  complete  digestion  of  the  yolk  in 
the  same  individuals  as  those  of  curve  A.  There  is  only  one  maximum 


48 


E.  J.  LUND 


sienpiAipul 


Fig.  6 Experiment  XXVI.  Curve  A represents  the  course  of  complete  extrusion  of  Chinese  ink  fed  to  fifty-four  indi- 
viduals; it  shows  two  maxima;  curve  B,  course  of  complete  digestion  by  the  same  individuals  as  of  curve  A;  curve  B 
has  only  one  maximum. 


Individuals 


RELATION  OF  BURSARIA  TO  FOOD 


49 


Fig.  7 Experiment  XXVII.  Curve  A,  course  of  extrusion  of  Chinese  ink  show- 
ing two  maxima  (cf.  fig.  6,  curve  A);  curve  B,  that  of  complete  digestion,  by  the 
same  individuals  used  in  curve  A,  of  extracted  yolk;  curve  C,  control:  course  of 
extrusion  of  ink  by  forty-eight  individuals  fed  ink  alone. 

in  this  curve,  and  this  comes  about  eighteen  hours  later  than  the  first 
maximum  of  curve  A and  at  about  the  same  time  as  the  second  maxi- 
mum of  B.  The  significance  of  the  second  maximum  in  curve  A is 
brought  out  in  the  following  analysis  of  the  two  curves  A and  B,  that 
is,  in  a quantitative  analysis  of  the  reactions  of  the  forty-eight  indi- 
viduals used  in  the  experiment.  All  the  individuals  which  at  any  of 
the  examinations  had  ink  and  yolk  present  in  the  same  vacuole,  or 
vacuoles,  were  recorded,  hence  we  have  a means  of  dividing  the  forty- 
eight  individuals  into  two  groups.  Group  I is  made  up  of  those  in 
which,  throughout  the  experiment,  yolk  and  ink  were  in  distinct  and 
separate  vacuoles,  while  Group  II,  includes  those  which  had  during 
part  or  all  of  the  time  one  or  more  vacuoles  which  contained  both  ink 
and  yolk  in  the  same  vacuole  or  vacuoles.  Now  we  have  the  data 
which  will  show  just  what  relation  ink  and  yolk  have  to  each  other  in 
the  cytoplasm  of  Bursaria,  and  what  the  reaction  of  the  cell  is  toward 
each  of  the  two  conditions  represented  by  the  composition  of  the  vacu- 
oles of  the  two  Groups  I and  II. 


THE  JOUBNAL  OF  EXPERIMENTAL  ZOOLOGY,  VOL.  16,  NO.  1 


50 


E.  J.  LUND 


Fig.  8 Analysis  of  curves  A and  B of  Experiment  XXVII,  figure  7.  7,  curve 

A,  course  of  complete  extrusion  of  ink  from  vacuoles  containing  only  ink;  curve 

B,  course  of  complete  digestion  of  extracted  yolk  in  vacuoles  ccftitaining  only 
yolk,  of  the  same  individuals  used  in  curve  A.  II,  Curve  A,  course  of  complete 
extrusion  of  ink  from  vacuoles  containing  both  ink  and  extracted  yolk;  curve 
B,  course  of  complete  digestion  of  extracted  yolk  from  the  same  vacuoles  in  the 
same  individuals  as  in  curve  A. 

We  may  plot  the  curve  of  extrusion  of  ink  in  group  I,  and 
the  curve  of  complete  digestion  of  the  same  individuals.  The 
curves  are  given  in  figure  8,  I.  The  same  was  likewise  done 
for  Group  II  (fig.  8,  II). 

Curve  A of  figure  8,  I,  (ink  and  yolk  in  separate  vacuoles) 
shows  now  only  one  early  extrusion  maximum  instead  of  two. 
Curve  B of  figure  8,  I,  is  lower  (owing  to  the  smaller  number  of 
individuals)  but  exactly  similar  to  B of  figure  7.  The  extrusion 
of  ink  from  a cell  which  has  its  yolk  and  ink  in  separate  vacuoles 
is  therefore  independent  of  the  presence  of  food  and  occurs  a 
long  time  before  digestion  of  the  food  is  completed.  The  curve 
of  extrusion  of  ink,  A,  figure  8,  II,  shows  now  only  one  maxi- 
mum and  this  corresponds  to  the  second  maximum  of  curve  A, 
figure  7,  and  is  practically  identical  with  curve  B of  complete 
digestion.  This  shows  then  that  whenever  ink  is  included  with 
food  in  the  same  vacuole  it  is  retained  until  its  accompanying 


RELATION  OF  BURSARIA  TO  FOOD 


51 


food  is  completel}^  digested,  because  the  maxima  of  the  curves 
A and  B of  II,  and  of  B,  I,  occur  at  the  same  time.  We  there- 
fore have  a demonstration  of  the  selective  extrusion  among  vac- 
uoles as  well  as  of  a process  of  selection  in  feeding  in  Bursaria. 
Similar  curves  may  be  worked  out  for  Sudan  III  or  powdered 
aluminium. 

SUMMARY 

1.  Bursaria  has  three  ways  of  rejecting  solid  particles,  as  shown 
by  the  paths  over  which  the  particles  are  passed.  These  are: 
(a)  the  path  of  total  rejection,  shown  by  particles  which  never 
enter  the  oral  apparatus;  (b)  the  path  of  rejection  of  large  par- 
ticles, this  being  a retracing  in  the  opposite  direction  of  the 
path  by  which  they  entered;  (c)  the  path  of  rejection  of  small 
particles,  which  leave  the  oral  pouch  by  way  of  the  base  of  the 
oral  sinus  and  are  passed  backward  over  the  ventral  side  of  the 
body  (fig.  1). 

2.  No  definite  path  is  followed  by  the  food  vacuoles  during 
digestion,  and  in  their  passage  through  the  cytoplasm.  Residues 
are  eliminated  from  a small  area  on  the  mid-dorsal  side  of  the 
cell. 

3.  Grains  of  fresh  hard  boiled  yolk  of  hens  egg,  when  prepared 
as  described  (page  8),  furnishes  a good  unit  of  measure  of  the 
food  taken,  and  an  easy  means  for  determining  the  factors  which 
come  into  play  in  the  process  of  feeding  in  Bursaria. 

4.  The  amount  of  food  eaten  and  the  rate  at  which  it  is  eaten 
depends  upon  the  physiological  state  of  the  cell  (defined  on  page 
10).  This  is  shown  to  be  true  for  fresh  and  for  fat-free  yolk, 
and  also  for  indigestible  substances  such  as  aluminium,  Sudan 
III,  Chinese  ink,  etc. 

5.  Change  in  the  physiological  state  of  the  cell  is  indicated 
by  the  change  in  the  total  amount  eaten  and  the  rate  of  feed- 
ing, under  the  same  conditions. 

6.  The  rate  of  feeding  is  not  affected  in  proportion  to  the 
concentration  of  the  yolk  suspension. 

7.  Mechanical  stimulation  decreases  the  rate  of  feeding  or 
inhibits  it,  roughly  in  proportion  to  the  degree  of  stimulation. 


52 


E.  J.  LUND 


8.  Rise  in  temperature  increases  the  rate  of  feeding  on  yolk. 

9.  Continuous  action  of  white  light  of  high  intensity  had  no 
detectable  effect  upon  feeding  on  yolk. 

10.  Feeding  may  continue  during  stimulation  by  a direct  elec- 
tric current  of  sufficient  intensity  to  control  the  direction  of 
movement  of  the  organism. 

11.  Bursaria  can  discriminate  between  and  select  non-toxic 
grains  of  yolk  from  among  toxic  ones.  Whether  or  not  Bursaria 
will  eat  yolk  grains  that  have  adsorbed  a soluble  substance  de- 
pends upon  (a)  the  steepness  of  the  effective  concentration  gra- 
dient of  the  dye,  between  the  grain  and  the  non-toxic  medium; 
and  this  in  turn  depends  upon  the  amount  of  dye  adsorbed 
which  is  subject  to  a reversible  adsorption;  (b)  the  specific  chem- 
ical properties  (^taste’?)  of  the  substance  adsorbed. 

12.  There  are  strong  reasons  for  believing  that  different  parts 
of  the  cell  are  affected  unequally  by  certain  toxic  substances,  and 
that  these  may  have  a specific  action  upon  the  selection  mecha- 
nism, causing  a more  definite  rejecting  reaction. 

13.  Yolk  which  has  adsorbed  a substance  which  is  insoluble 
in  water  (Sudan  III)  is  eaten  as  readily  as  fresh  unstained  yolk. 

14.  Bursaria  has  the  power  of  selective  extrusion  among  vacu- 
oles each  containing  different  substances  eaten  at  the  same  time; 
vacuoles  containing  indigestible  substances  are  soon  extruded, 
while  those  containing  food  are  retained.  If  fat-free  yolk  is 
present  in  the  same  vacuole  along  with ’the  indigestible  sub- 
stance, then  the  latter  is  retained  until  digestion  of  the  enclosed 
yolk  has  run  its  usual  course. 

LITERATURE  CITED 

Greenwood,  M.  1894  On  the  constitution  and  mode  of  formation  of  food 
vacuoles  in  Infusoria,  etc.  Philos.  Transact.  Roy.  Soc.,  London,  voL 
185,  B,  p.  355. 

Metalnikow,  S.  1912  Contributions  a I’etude  de  la  digestion  intracellulaire 
chez  les  Protozoaires.  Arch.  Zool.  Exper.,  tome  9,  pp.  373-499. 
Schaeffer,  Asa  Arthur  1910  Selection  of  food  in  Stentor  coeruleus  (Ehr.) 
Jour.  Exper.  Zool.,  vol.  8,  pp.  75-132. 


BIOGRAPHY 


I,  Elmer  J.  Lund,  second  son  of  Cecelia  and  Peter  A.  Lund,  was 
born  December  13,  1884,  near  Springfield,  Redwood  County, 
Minnesota.  My  early  education  was  obtained  in  the  public 
Schools  of  Olivia,  Renville  County,  Minnesota,  and  I graduated 
from  the  high  school  at  that  place  in  1906.  In  the  fall  of  that 
year  I entered  Hamline  University,  where  I graduated  with  the 
degree  of  Bachelor  of  Philosophy,  in  1910.  During  the  summer 
of  1910  I was  a member  of  the  Zoological  Expedition  of  the  Johns 
Hopkins  University  to  Jamaica.  After  returning  I entered  the 
Johns  Hopkins  University  as  a graduate  student  in  Zoology,  with 
Botany  and  Physical  Chemistry  as  subordinate  subjects.  . During 
my  first  year  I was  also  student  assistant  in  Zoology.  In  the 
summer  of  1911  I carried  on  research  in  Zoology,  and  studied 
Physics,  at  the  University  of  Chicago.  I published  in  the  Journal 
of  Experimental  Zoology  for  1911  a paper  entitled  ''On  the  Struc- 
ture, Physiology  and  Use  of  Luminous  Organs,  with  Special 
Reference  to  the  Lampyridae.  ’’ 

During  the  years  1911-1914  I have  held  the  Adam  T.  Bruce 
Fellowship  in  Biology  at  the  Johns  Hopkins  University.  In  the 
summers  of  1912  and  1913  I have  been  instructor  in  Zoology  at  the 
Alarine  Biological  Laboratory  at  Woods  Hole,  Massachusetts. 


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